Christina M. Mikulski

Optimization of solid-phase extraction and liquid chromatography–tandem mass spectrometry for the determination of domoic acid in seawater, phytoplankton, and mammalian fluids and tissues ☆

We previously reported a solid-phase extraction (SPE) method for determination of the neurotoxin domoic acid (DA) in both seawater and phytoplankton by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose of sample desalting without DA pre-concentration. In the present study, we optimized the SPE procedure with seawater and phytoplankton samples directly acidified with aqueous formic acid without addition of organic solvents, which allowed sample desalting and also 20-fold pre-concentration of DA in seawater and phytoplankton samples. In order to reduce MS contamination, a diverter valve was installed between LC and MS to send the LC eluant to waste, except for the 6-min elution window bracketing the DA retention time, which was sent to the MS. Reduction of the MS turbo gas temperature also helped to maintain the long-term stability of MS signal. Recoveries exceeded 90% for the DA-negative seawater and the DA-positive cultured phytoplankton samples spiked with DA. The SPE method for DA extraction and sample clean-up in seawater was extended to mammalian fluids and tissues with modification in order to accommodate the fluid samples with limited available volumes and the tissue extracts in aqueous methanol. Recoveries of DA from DA-exposed laboratory mammalian samples (amniotic fluid, cerebrospinal fluid, plasma, placenta, and brain) were above 85%. Recoveries of DA from samples (urine, feces, intestinal contents, and gastric contents) collected from field stranded marine mammals showed large variations and were affected by the sample status. The optimized SPE-LC-MS method allows determination of DA at trace levels (low pg mL(-1)) in seawater with/without the presence of phytoplankton. The application of SPE clean-up to mammalian fluids and tissue extracts greatly reduced the LC column degradation and MS contamination, which allowed routine screening of marine mammalian samples for confirmation of DA exposure and determination of fluid and tissue DA concentrations in experimental laboratory animals.

Comparative analysis of bacterioplankton assemblages from Karenia brevis bloom and nonbloom water on the west Florida shelf (Gulf of Mexico, USA) using 16S rRNA gene clone libraries.

The brevetoxin-producing dinoflagellate, Karenia brevis, forms nearly annual blooms off the Florida west coast, severely impacting the regions ecology and economy. Bacteria are often cited as either promoting or interfering with the development of algal blooms, and thus a detailed study of the bacterioplankton assemblages associated with K. brevis was undertaken. We developed sixteen 16S rRNA gene clone libraries from K. brevis bloom and adjacent nonbloom water to determine the bacterial groups present and assess the influence of K. brevis cell number and/or depth on bacterioplankton community composition. Most notably, bacterial groups such as Rhodobacterales (Alphaproteobacteria) and Cytophagales/Sphingobacteriales (Bacteroidetes), reported previously to be associated with other harmful algal species, were often abundant in the presence of K. brevis. Cyanobacteria frequently dominated surface samples containing no detectable K. brevis, consistent with earlier work suggesting that these photosynthetic organisms may be important in promoting the proliferation of these blooms by conditioning the water. Moreover, differences in the abundance/diversity of traditionally more rare and often undocumented phylogenetic groups (e.g. Betaproteobacteria, Deltaproteobacteria, Chloroflexus, Firmicutes) were apparent in bloom vs. nonbloom water. This is the first study to document the association of these phylogenetic groups with natural K. brevis populations and suggests a potential role for these microorganisms in K. brevis bloom dynamics.

Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay: Perspectives from targeted and adaptive sampling

Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo-nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.