Location

Water collection sites in the northern Gulf of Mexico, particularly at the mouth of Mobile Bay and Little Lagoon, AL.

Water Collection

Briefly, water was collected from the field using a 5-gallon bucket, pre-screened with a 200 µm nitex mesh, and gently poured into 10-20 L carboys and kept in the dark until returning to the laboratory for same-day processing.

Terminology

dDA – dissolved Domoic Acid
pDAa – particulate Domoic Acid (algal fraction)
pDAOP – particulate Domoic Acid (bound to organic polymers)
cDA – Domoic Acid in copepods
POC – Particulate Organic Carbon

Field-simulation experiments

Field water used for grazing experiments was collected during spring and summer (2017, 2018) from designated monitoring sites at Little Lagoon (Gulf Shores, Alabama, USA). The water was prefiltered with a 200 µm mesh, gently poured into carboys, and kept in the dark until the start of the laboratory experiment. Laboratory-reared adult Acartia tonsa, with no prior exposure to DA, were provided by the University of Southern Mississippi Gulf Coast Research Laboratory’s Thad Cochran Marine Aquaculture Center and starved for 24 hours prior to experiment initiation.

Initial samples for Pseudo-nitzschia abundance and DA were collected. For cell abundance, 50 mL of seawater was preserved with 2 mL of Bouin’s solution and stored at 4°C. A Sedgewick rafter slide was used to count cells in a 1 mL subsample. DA was measured in two forms, dDA and pDA. Seawater was filtered using a 25 mm glass fiber filter and 30 mL of filtrate was collected in a polypropylene conical tube and stored at -20°C for later analysis of dDA. pDA was sampled by filtering 100 mL of seawater under low vacuum through a 25 mm glass fiber filter and stored in a cryovial at -20°C.

The experimental design consisted of four treatments, each containing triplicate 1-L polycarbonate bottles. Two treatments contained seawater filtered through a 0.2 µm polycap filter (as described above); these treatments tested whether copepods could assimilate dDA through the proposed organic polymer-bound pathway. The remaining treatments contained seawater with a natural phytoplankton community that was concentrated by a factor of three, using a 20 µm mesh. After the bottles were filled with the appropriate water, 30 copepods were added to the necessary treatments and the experiments started. After 24 hours the copepods were collected on a 200 µm screen, gently rinsed with filtered seawater, placed in fresh filtered artificial seawater, and allowed to evacuate their guts for ~1 hour. Afterwards, copepods were once again screened and rinsed three times, and then stored in a cryovial at -20°C until analysis.

Liquid chromatography-mass spectrometry method for domoic acid quantification

LC-MS sample preparation followed was modified from Wang et al. (2012) for the determination of dDA, pDA, pDAOP and cDA. The samples for DA determination were cleaned and concentrated using Bond Elut LRC - C18, 200 mg, solid-phase extraction (SPE) columns from Agilent Technologies. For dDA, 30 mL seawater samples were filtered using a 47 mm glass fiber filter; the filtrate was collected and acidified with formic acid to yield a 0.2% final solution. SPE columns were conditioned with one column volume of HPLC-grade methanol followed by one column volume of HPLC-grade water. Samples were then loaded on the SPE column and filtered at ~1 mL min-1 using a vacuum manifold, followed by 10 mL of 0.2% formic acid as a rinse for the sample tube and SPE column. The SPE column was then allowed to go dry and was eluted with 1.5 mL of 20 mM ammonium acetate in 50% methanol (pH 8) and collected in a glass tube. The tubes were centrifuged for 5 minutes at ~1300 x g, supernatant was transferred into an LC vial with a Pasteur pipette, and stored at 4°C until further analysis. For pDAa 100 mL of seawater were filtered through a 5 µm polycarbonate filter and stored in a 50 mL polypropylene tube at -20°C. Similarly, for pDAOP 150 mL of seawater was filtered through a pre-combusted 25 mm glass-fiber filter and stored at -20°C. Prior to concentration and clean-up for pDA, pDAOP, and cDA, the filters were submerged in 2 mL of 80% methanol and sonicated to ensure cells and copepods were lysed. Sonication pulses were done for a total of 45 seconds (5 seconds on/off) on a Sonics Materials Ultrasonic Processor (model - VCX 130) at 75% power. Subsequent clean-up using the SPE column is the same as for the dDA samples.

An ultra-performance liquid chromatography (UPLC) – tandem mass spectrometry (MS) system was used for the quantification of DA.The LC-MS system consisted of Acquity UPLC system (Waters, Milford, MA) coupled to a 5500 QTRAP triple quadrupole / linear ion trap mass spectrometer equipped with a TurboIonSpray interface (Sciex, Foster City, CA, USA). The analytes were separated on a Luna C18 (2), 2.0 x 100 mm column (Phenomenex, Torrance, CA, USA) with column temperature held at 40ºC. The mobile phase was water (A) and 95% aqueous acetonitrile (B) with 0.1% formic acid additive and the flow rate was 0.4 ml/min. Gradient program was: 5% B for 3 min, linear gradient to 60% B at 10 min, 95% B at 10.1 min, hold at 95% B for 2 min. MS was operated in positive ion mode. Ion spray voltage was 5 kV and declustering potential was 80 V. Gas parameter settings were: nebulizer gas, 50 psi; turbo gas, 50 psi at 500ºC; curtain gas, 20 psi; and collision gas, medium setting. The collision energy applied was 25eV. The transitions used for selected reaction monitoring were m/z 312→266, 193, 220. The transition m/z 312→266 was used for quantitation.

For organic polymer formation and sorption of DA results and methodology see https://www.bco-dmo.org/dataset/808280.