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A fluorometric assay was developed to semi-quantify co-purified polyketide prymnesins-1 and -2 (PPs) from Prymnesium parvum cultures. Evaluations performed throughout the growth cycle of 5 practical salinity unit (PSU) cultures detected relatively 8–10 × more PPs in the culture medium (exotoxins) than in cells (endotoxins). The [exotoxin] remained stable and relatively low until post-log growth, when they increased significantly. However, on a per-cell basis, [exotoxin] declined throughout log phase and subsequently increased dramatically during late- and post-log phases. The [endotoxin] remained stable until late- and post-log phases, when it achieved its highest level before declining sharply. Shaking cultures of strains from Texas, South Carolina and the United Kingdom displayed dramatically different [exotoxin] during post-log decline. Cultures adapted to 30 PSU had significantly lower [exotoxin] over the course of cultivation than those grown at 5 PSU. Phosphate limitation enhanced [exotoxin] on a per-cell basis, especially in late- and post-log cultures. Media containing streptomycin exhibited a ∼20% increase in [exotoxin] in post-log cultures vs. control treatments, but it had only negligible effects on endotoxin levels. Brefeldin A had minimal effects on [exotoxin], suggesting that the presence of PPs in the medium may be largely derived from cell lysis or some other passive means. [ABSTRACT FROM AUTHOR]

Abstract: Known as Golden Algae in popular media, the harmful algal bloom causing organism Prymnesium parvum secretes increased amounts of toxic chemicals called prymnesins when stressed, resulting in major fish kills in Texas. Although many options exist for mitigation of blooms, a feasible protocol for control of blooms on large-scale impoundments has yet to be identified. Chemical control of P. parvum using six different enzyme inhibiting aquatic herbicides was explored in laboratory experiments. Of the six chemicals screened, one (flumioxazin) was selected for further study due to a significant decrease in P. parvum cell numbers with increasing chemical concentration. It was applied to natural plankton communities during in-situ experiments (Lake Granbury, Texas). The first experiment was conducted during a period of P. parvum bloom initiation (March) and the second experiment conducted during a post bloom period (April). Experiments were carried out in 20 L polycarbonate carboys covered in 30% shade cloth to simulate natural light, temperature and turbulence conditions. Through cell counts via light-microscopy, the chemical flumioxazin was found to cause significant decreases in P. parvum, but no significant differences in zooplankton abundance during the period of bloom initiation. However, significant decreases in adult copepods were observed during the post bloom period, with no significant decreases in P. parvum most likely due to decreased light penetration and inhibition of the photosensitive mode of action. [Copyright &y& Elsevier]

Abstract: Within the last two decades, Prymnesium parvum (golden algae) has rapidly spread into inland waterways across the southern portion of North America and this organism has now appeared in more northerly distributed watersheds. In its wake, golden algae blooms have left an alarming trail of ecological devastation, namely massive fish kills, which are threatening the economic and recreational value of freshwater systems throughout the United States. To further understand the nature of this emerging crisis, our group investigated the chemical nature of the toxin(s) produced by P. parvum. We approached the problem using a two-pronged strategy that included analyzing both laboratory-grown golden algae and field-collected samples of P. parvum. Our results demonstrate that there is a striking difference in the toxin profiles for these two systems. An assemblage of potently ichthyotoxic fatty acids consisting primarily of stearidonic acid was identified in P. parvum cultures. While the concentration of the fatty acids alone was sufficient to account for the rapid-onset ichthyotoxic properties of cultured P. parvum, we also detected a second type of highly labile ichthyotoxic substance(s) in laboratory-grown golden algae that remains uncharacterized. In contrast, the amounts of stearidonic acid and its related congeners present in samples from recent bloom and fish kill sites fell well below the limits necessary to induce acute toxicity in fish. However, a highly labile ichthyotoxic substance, which is similar to the one found in laboratory-grown P. parvum cultures, was also detected. We propose that the uncharacterized labile metabolite produced by P. parvum is responsible for golden algae''s devastating fish killing effects. Moreover, we have determined that the biologically-relevant ichthyotoxins produced by P. parvum are not the prymnesins as is widely believed. Our results suggest that further intensive efforts will be required to chemically define P. parvum''s ichthyotoxins under natural bloom conditions. [Copyright &y& Elsevier]

Abstract: In marine and estuarine conditions, pH-dependent aquatic toxicity associated with Prymnesium parvum has been previously demonstrated with greater toxicity observed at higher pH. Recent research by our group extended such observations to inland waters in Texas, USA. Because prymnesins (prymnesin-1, prymnesin-2) represent the only P. parvum toxins reported in the peer-reviewed literature, we hypothesized that P. parvum toxins may behave like weak bases in surface waters due to the presence of a primary amine. Employing a widely accepted computational model (ACD/Labs), we predicted physicochemical properties to support interpretation of experimental findings, specifically focusing on the amine-containing hydrophobic portion of these molecules. Using different computational models, alternative pK a values for prymnesins have been proposed by others. Herein, additional examination of our proposed hypothesis identifies the importance of considering subtle changes in ionization state on the lipophilicity, bioavailability (e.g., log D, log Dlip−water, Bioconcentration factor), and aquatic toxicity of ionizable chemicals over an environmentally-relevant pH gradient. If prymnesins represent causative toxins released by P. parvum as previously reported or if new toxins are identified in the future, a closer evaluation of our findings further highlights the importance of considering site-specific pH during environmental assessment and management efforts for P. parvum in inland waters. [Copyright &y& Elsevier]

Abstract: The harmful algal bloom species Prymnesium parvum has caused millions of dollars in damage to fisheries around the world. These fish kills have been attributed to P. parvum releasing a mixture of toxins in the water. The characterized toxins, reported as prymnesin-1 and -2, have structural similarities consistent with other known ionizable compounds (e.g., ammonia). We investigated whether pH affects the toxicity of P. parvum under conditions representative of inland Texas reservoirs experiencing ambient toxicity from bloom formation. We evaluated pH influences on toxicity in laboratory and field samples, and modeled the physicochemical properties of prymnesins. Aquatic toxicity to a model fish and cladoceran was reduced by lowering pH in samples obtained from reservoirs experiencing P. parvum blooms; similar observations were confirmed for experiments with laboratory cultures. A pKa value of 8.9 was predicted for the prymnesins, which suggests that ionization states of these toxins may change appreciably over surface water pH of inland waters. These findings indicate that ionization states of toxins released by P. parvum may strongly influence site-specific toxicity and subsequent impacts to fisheries. Consequently, these results emphasize the importance of understanding processes that affect pH during P. parvum blooms, which may improve predictions of ambient toxicity. [Copyright &y& Elsevier]

Abstract: A reconsideration of the pKa values proposed by Valenti et al. (Theodore W. Valenti et al., A mechanistic explanation for pH-dependent ambient aquatic toxicity of Prymnesium parvum carter. Toxicon (2010) 55, 990–998) suggests that the ionization status of the C-14 amino group in prymnesins will not substantially influence the toxicity of these compounds over a pH range of 6.5–8.5. A revised pKa value and ionization state distribution for the prymnesins in aqueous systems is proposed. [Copyright &y& Elsevier]