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. Author manuscript; available in PMC: 2015 Oct 2.
Published in final edited form as: Harmful Algae 2002 (2002). 2004;10:153–154.

Brevetoxin Degradation and By-Product Formation via Natural Sunlight

Ron C Hardman 1, William J Cooper 2, Andrea J Bourdelais 2, Piero Gardinali 3, Daniel G Baden 2
PMCID: PMC4591915  NIHMSID: NIHMS187078  PMID: 26436141

Abstract

We investigated the effects of solar radiation on brevetoxin (PbTx2). Our findings suggest that natural sunlight mediates brevetoxin (PbTx2) degradation and results in brevetoxin by-product formation via photochemical processes.

Introduction

Harmful algal blooms (HAB) of the dinoflagellate Karenia brevis are an annual concern in the US, affecting all areas of the Gulf of Mexico (GOM). Karenia brevis produces brevetoxin (PbTx), responsible for mass morbidity and mortality among marine wildlife such as fish, birds and marine mammals and the causative agent of Neurotoxic Shellfish Poisoning (NSP) in humans (Forrester et al., 1977; Baden, 1983; O’Shea, 1991; Bossart et al., 1998; Wilson et al., 1999). Brevetoxins are among nature’s most potent naturally occurring voltage gated sodium channel (VGSC) agonists, pharmacologically active in nanomolar concentrations. There are at least 10 distinct brevetoxins which vary in metabolic stability, pharmacological activity and potency (Gawley et al., 1995; Rein et al., 1994), characteristics which may be modified via metabolic and environmental processes as well as (as this paper will suggest) photo-chemical processes. While the impacts of brevetoxin(s) upon marine wildlife and humans are well known, a complete etiology of brevetoxicosis remains poorly defined and the transport and fate of brevetoxin in the environment has not been well characterized. In this study the effect of solar radiation on brevetoxin degradation was explored.

Materials and Methods

Samples (100 mL) of seawater, sea-surface microlayer and deionized water spiked with 150 μg to 450 μg of brevetoxin (PbTx2) were subjected to solar radiation (both natural and simulated sunlight) and dark conditions for varying periods of time (2 to 96 hours). Following exposures the samples were extracted with ethyl acetate (1:1) and the organic layer dried before resuspending in methanol. The suspended samples were filtered (0.2 μ nylon membrane filter) and analyzed via HPLC. Photochemical byproducts were further assayed via HPLC-MS, 1H-NMR and enzyme linked immunosorbent assay (ELISA). HPLC grade methanol, acetone, ethyl acetate and deionized and HPLC grade water were used in the extraction (described above) and HPLC analyses. A Hewlett-Packard 1100 HPLC (using Chem-station software) was used with a 5 μM C18 reverse phase column (Agilent Technologies) and a diode array detection wavelength of 215 nm. Either isocratic or gradient mobile phases of 85:15 MeOH;H2O (flow of 1.4 mL/min.) were employed in sample analysis and byproduct isolation. Artificial sunlight exposures were made using a Spectral Energy LPS 256 SM and LH 1153 solar simulator generating a solar spectrum from 300 nM to 700 nM. Measurements of the natural sunlight at the exposure location (Wilmington, N.C., noonday sun on June 21st) were PAR-1670 μE/m2/sec, UVA-3060 μW/cm2 and UVB-290 μW/cm2. The measured solar simulator spectra was PAR-1830 μE/m2/sec, UVA-4590 μW/cm2 and UVB-820 μW/cm2.

Results

The HPLC profiles of PbTx2 spiked aqueous solutions subjected to solar radiation differed markedly from the PbTx2 spiked aqueous solutions kept under dark conditions (Fig. 1). At least 18 chemical entities were detected in the solar exposed samples which were not detected, or present in markedly lower concentrations, in samples stored under dark conditions. These photochemical by-products were consistently observed in each assay (n = 17). Figure 1 plots the average profile change of the solar exposed versus dark conditions samples over a 24 hr period (not all by-products are shown for clarity). The plots are based on peak area determined by HPLC chromatograms. Semi-quantitative analysis indicates brevetoxin (PbTx2) concentrations were reduced ~35% after 24 hrs of exposure to natural sunlight. The photochemical by-products (denoted in Fig. 1 by ByPrd 3.2, etc.) were found to represent from 2% to 13% of the total material in the sample after 24 hrs of exposure. In contrast the average PbTx2 degradation was ~3% under dark conditions. Subsequent experiments (n = 2) of 96 hr natural sunlight exposures yielded profiles where by-products represented ~75% of the total material in the sample and PbTx2 represented ~25% of the total material in the sample. Thus, brevetoxin (PbTx2 spike) concentrations were found to decrease while photochemical by-products were found to increase when samples were subjected to solar radiation. Four of the photochemical by-products were isolated in adequate quantities and purity to permit preliminary characterization. Each of the four products demonstrated a positive enzyme linked immunosorbent assay (ELISA) for brevetoxin. 1H-NMR spectra for each of the four by-products suggested similarities to known brevetoxin standards (PbTx2 and PbTx9). HPLC-MS analyses of the photoproducts suggests the majority of these brevetoxin derivatives may be novel; most (19 of 21) of the by-product masses detected did not correlate to any known brevetoxins or brevetoxin metabolites in published literature. The major ions detected were mz (m + h) = 577,579, 621, 848, 877, 885, 895, 899, 904, 912, 913, 914, 927, 930, 944, 949, 958, 962, 972, 973, 981. In summary, the photo-chemical by-products/derivatives are of brevetoxin (PbTx2) origin and those analyzed demonstrate structural similarities to the PbTx2 parent compound.

Figure 1.

Figure 1

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Average PbTx2 profile changes over 24 hr exposure to solar radiation and dark conditions (*note dual axis). Photochemical by-products (not all shown for clarity) are denoted by “ByPrd” (bars). The Y2 axis is for the PbTx2 spike concentration (~35% reduction), the Y1 axis is for the concentration of the photoproducts (2% to 13% increase in solar exposed). PbTx2 and by-products shown as a percentage of total material in the sample. In the solar exposed samples, photoproduct concentrations were found to increase while PbTx2 concentrations declined with time. The samples subjected to dark conditions show markedly less activity. There was no difference in PbTx2 degradation or by-product formation under natural and simulated sunlight.

Discussion

Results from this work suggest natural sunlight plays an important role in brevetoxin (PbTx2) degradation in the natural environment. Our findings show solar radiation mediates PbTx2 degradation and photochemical by-product formation via first order photochemical processes. Approximately 18 PbTx2 degradation products, the majority of which appear to be novel, were found. These photo-products were not observed to degrade over the time frames studied and appear to be more stable than the parent compound, PbTx2; however, no similar data exist for the stability of these photoproducts in the natural environment. The biological activity of these photochemical by-products is not yet known; work on their biological activity is currently being conducted at Duke University.

During the course of this study Welker and Steinberg (1999) reported the indirect photolysis of microcystins, potent cyanobacterial hepatotoxins. Their findings indicated that, unlike the results here, photodegradation was via indirect photolysis and required the presence of humic substances. More recently it was shown that domoic acid, a potent neurotoxin produced by the diatom Pseudo-nitzschia multiseries, also undergoes photodegradation, forming various geometrical isomers as well as decar-boxylated products (Campbell et al., 2002). Hence, it is apparent that solar radiation can play a role in the degradation of a variety of aquatic toxins. These findings will hopefully augment our understanding of bloom dynamics and marine biotoxin speciation in natural waters.

Acknowledgments

This research was funded in part by a Glaxo-Wellcome Foundation Oceans and Human Health Fellowship. We thank Susan Campbell, Jodi Lamberto, Allison Weidner and Karl Jacocks of the University of North Carolina at Wilmington for their assistance and algal toxin production.

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