The CBA-N2a detectable CTX concentrations in lionfish from two of the sampling locations are shown as C-CTX-1 eq. values in ppb.

<p>The number of fish analyzed at each location was 11 for the British Virgin Islands (BVI) and 5 for the Florida Keys. C-CTX-1 was confirmed by LC-MS/MS (denoted by an *) in six of the samples from BVI and one from Florida Keys. The horizontal dashed line indicates the FDA guidance level of 0.1 ppb for C-CTX-1 eq.</p

Ciguatoxin concentrations in lionfish reported from two previous studies done in the Lesser Antilles and US Virgin Islands determined by CBA-N2a compared to the CTX estimates found in this study.

<p>ND = CTX activity not detected.</p

Range of reported C-CTX-1 concentrations for Caribbean and Gulf of Mexico fish other than lionfish.

<p>Range of reported C-CTX-1 concentrations for Caribbean and Gulf of Mexico fish other than lionfish.</p

LC-MS/MS chromatogram for confirmation of C-CTX-1.

<p>The chromatogram shows the three characteristic confirmatory ion transitions (<i>m/z</i> 1123.6 > 1105.6, 1123.6 > 1087.6, and 1123.6 > 1069.9) and the retention time (4.8 min) of a C-CTX-1 standard and in lionfish.</p

Map showing distribution of sampling sites where fish were collected.

<p>Sample sites located within each location are identified as “n”.</p

Location based fluorescent receptor binding assay (RBA_F) results list the date of sampling, number of lionfish screened and the number of positive samples.

<p>Following the RBA_F screening, all the positive samples (n = 30) plus a subset of negative samples (n = 24) were next analyzed using CBA-N2a and LC-MS/MS. The negative samples identified by RBA_F were also negative when measured by CBA-N2a and LC-MS/MS. CBA-N2a and LC-MS/MS confirmed that a subset of the samples screened contained measurable levels of C-CTX-1. Location abbreviations FGBNMS and USVI stand for Flower Garden Banks National Marine Sanctuary and United States Virgin Islands, respectively.</p

Time Series of RBA_(F) Reaction.

<p>RBA_(F) standard curves of percent (%) binding versus P-CTX-3C (g mL^-1) where the standards were allowed to incubate with the synaptosomes for 1.5 (open circle), 3.0 (open triangle) and 4.0 h (open square).</p

LC-MS/MS Confirmation of Ciguatoxins.

<p>LC-MS/MS chromatogram showing the retention time (4.76 min) and the three characteristic ion transitions (1123.6 > 1105.6, 1123.6 > 1087.9, and 1123.6 > 1069.6) of a C-CTX-1 standard and in a lionfish sample.</p

CBA-N2a Ciguatoxin Standard Curves.

<p>CBA-N2a standard curves for P-CTX-3C (closed circle) and P-CTX-1 (open triangle) with percent (%) viability of Neuro-2a plotted against toxin concentration (g mL^-1). Percent cell viability is defined as the survival ratio of cells treated with and without ouabain and veratridine. Error bars represent ± standard deviations of multiple standard curves. The number of curves included is given as (N).</p

RBA_(F) and RBA_(R) Linear Standard Curves.

<p>Lower ciguatoxin concentrations found in fish surveyed in this study were accurately measured using a linear standard curve consisting of 0.10, 0.25, 0.50 and 1.0 ppb P-CTX-3C. Results were equivalent for the RBA_(F) (open circle; solid line) and RBA_(R) (closed triangle; dashed line) (t-test, P = 0.982).</p