3.1. Experimental exposure of omnivorous fish to toxic shellfish
A subset of hard clams used in the exposure studies was analyzed by LC-MS prior to exposures and was found to contain PbTx-3 and metabolites. Brevetoxin concentration by ELISA was 1800 ng g−1, and toxicity assessed by mouse bioassay was 30 mouse units (MU) per 100 g. No brevetoxins were detected by ELISA in the control clams.
While feeding toxic hard clams for 2 consecutive weeks, none of the pinfish or croakers died or exhibited any obvious signs of adverse effects. For comparison with the planktivorous fish exposures, toxin concentrations measured in liver and remaining viscera were averaged by tissue weight and are referred to as viscera. Higher concentrations of brevetoxins were measured on day 14 compared to day 7 of the exposures (), with the maximum average levels in pinfish (1412±151 ng g−1 in muscle and 2233±442 ng g−1 in viscera, n = 3) exceeding those measured in croakers (955±73 ng g−1 in muscle and 1990±166 ng g−1 in viscera, n = 3). The highest levels measured in an individual fish were 2675 ng g−1 in viscera and 1540 ng g−1 in muscle in a pinfish on the final day of the exposure period (day 14). When fish were switched to a diet of nontoxic clams, brevetoxin concentrations dropped but remained detectable in both muscle and viscera after 2 weeks of feeding on the nontoxic diet. Brevetoxins were not detected in fish on day 0 (before exposure) or in any of the control fish.
Fig. 1 Brevetoxin concentrations measured in muscle and viscera of pinfish (L. rhomboides) and croakers (M. undulatus) fed brevetoxin-contaminated clams (day 1–14) followed by noncontaminated clams (day 15–28). Error bars indicate standard deviation (more ...)
3.2. Experimental exposure of planktivorous fish to K. brevis culture
During the exposure of juvenile striped mullet to K. brevis cultures (500–4000 cells ml−1), microscopic observations of the exposure media at 6 and 24 h verified that K. brevis cells remained intact. ELISA analyses of the exposure media after 6 and 24 h confirmed that greater than 95% of brevetoxin measured in the water was associated with the K. brevis cells (). Under these conditions, all fish survived and quickly accumulated brevetoxins in both muscle and viscera. After 6 h, brevetoxins were measurable in all fish with concentrations ranging from 25 to 231 ng g−1 in muscle and 120–335 ng g−1 in viscera (data not shown). Brevetoxin concentrations in fish tissues increased with increasing K. brevis cell densities and with the duration of exposure—reaching an average of 333 ng g−1 in muscle and 616 ng g−1 in viscera after 24 h (). In viscera, this increase was linear with increasing cell concentration (r2 = 0.998), while in muscle the increase was only linear up to 2000 cells ml−1 (r2 = 0.91), with no further increase at the highest cell density. Brevetoxins were not detected in any of the control fish.
Fig. 2 (a) Brevetoxin concentrations in muscle and viscera of juvenile striped mullet after 24 h exposure to K. brevis cell cultures at indicated cell densities, and total and dissolved concentrations of brevetoxins measured in exposure media. (b) Time to the (more ...)
In parallel experiments, the ichthyotoxicity of dissolved brevetoxins was confirmed when K. brevis cells were lysed by sonication before addition to the aquaria (). In this case, the fish did not accumulate toxins but died quickly (7–80 min), and the time to death correlated with the number of lysed cells (r2 = 0.99). In both experiments, the total brevetoxin concentrations in the aquaria were nearly identical (ranging from 8 to 66 ng ml−1) and were proportional to the initial K. brevis cell densities ().
3.3. Field results
Maximum K. brevis cell densities observed in St. Joseph Bay each month from January 2005 through December 2006 are shown in . No K. brevis cells at bloom densities were observed in samples collected in St. Joseph Bay between January and July 2005; and only background levels were present in a few of the samples collected in April and May 2005. In August 2005, a southwest Florida K. brevis bloom was transported into the Florida panhandle, and elevated cell densities were first observed in St. Joseph Bay in a sample collected on August 31, 2005. In September 2005, K. brevis cell concentrations in the bay reached as high as 2.5 million-cells l−1 (). K. brevis cell densities remained elevated in the bay until late October. In mid-November 2005, maximum densities of 5300 K. brevis cells l−1 were observed. Subsequent to this date, no K. brevis were found until May 2006, when very low levels (maximum of 4000 cells l−1) were noted at only a few sites. Brevetoxin concentrations measured in St. Joseph Bay water samples ranged from <1 to 80 μg PbTx l−1 during the red tide bloom in September 2005, and were often measurable but low (typically <1 μg l−1) in the absence of a K. brevis bloom ().
Maximum Karenia brevis cell densities and brevetoxin concentrations measured in seawater samples collected throughout St. Joseph Bay between January 2005 and December 2006.
From February 2005 to November 2006, 184 fish representing 38 species of varying diets and trophic groups were collected from St. Joseph Bay and analyzed for brevetoxins. Species collected and maximum brevetoxins concentrations measured in muscle, liver and stomach contents are listed in , which includes 101 fish collected during the spring of 2004. Species included small planktivorous thread herring (Opisthonema oglinum) and sardines (Harengula jaguana), large piscivorous bluefish (Pomatomus saltatrix) and seatrout (Cynoscion nebulosus), as well as demersal and benthic omnivorous species such as pinfish (L. rhomboides) and catfish (Ariopsis felis).
Maximum brevetoxin concentrations measured in fish collected from St. Joseph Bay between March 2004 and November 2006
The average brevetoxin concentrations measured by ELISA in muscle, liver and stomach contents for each collection are shown in , and the prevalence of brevetoxins, expressed as the percentage of brevetoxin-positive samples (by ELISA) of each tissue type, is shown in . Although no K. brevis bloom was noted in the bay until September 2005, brevetoxins were detected in more than 69% of fish collected in February and June 2005 (). In general, average levels were highest during and immediately following the red tide in September and November 2005, after which they slowly decreased ().
Fig. 4 Average concentrations of brevetoxins measured in fish (all species) collected throughout St. Joseph Bay between 2004 and 2006. In 2005 and 2006, fish collections were performed during 2–3 day sampling trips. Error bars indicate standard deviation. (more ...)
Fig. 5 Prevalence of brevetoxin contamination in fish (all species) collected throughout St. Joseph Bay between 2004 and 2006. Prevalence is expressed as the percentage of fish containing detectable levels of brevetoxins (>5–10 ng g−1 (more ...)
In February 2005, no toxin was measured in the muscle of any fish. In June, 10% of the fish tested contained low brevetoxin levels (13–20 ng g−1) in the muscle. During the K. brevis bloom in September 2005, brevetoxins were measurable in the muscle of 96% of the fish tested, with concentrations ranging from 9 to 581 ng g−1. In November 2005 after the K. brevis bloom, brevetoxin was found in the muscle of 88% of the fish collected (n = 17), with a maximum concentration of 116 ng g−1 measured in a gag grouper (Mycteroperca microlepis). By May 2006, both the proportion of positive muscle samples (64%, n = 23) and the concentrations measured in the muscle had decreased. With the exception of one hardhead sea catfish (A. felis), brevetoxin was not found in the muscle of any fish collected in August or November 2006. Overall, the highest brevetoxin concentration we measured in muscle (581 ng g−1) was from a Spanish sardine (Sardinella aurita) collected during the September 2005 red tide.
Liver samples consistently yielded the greatest proportion of brevetoxin-positive results ( and ). In February 2005, brevetoxin concentrations in fish livers ranged from not detectable (<5–10 ng g−1) in 30% of the fish collected to 81 ng g−1. Average liver brevetoxin concentrations increased in each collection reaching peak levels in November 2005, just after the red tide ended. The highest liver concentration measured was 16,483 ng g−1 in a red snapper (Lutjanus campechanus) collected in November 2005. In May 2006, the average concentration in the livers had decreased but was still high (598 ng g−1), and brevetoxin was detected in 100% of the livers tested. In August and November 2006, the averages continued to decline, but brevetoxin was still found in the livers of more than half of the fish collected.
Brevetoxin concentrations in stomach contents displayed the greatest variability. In February and June 2005, brevetoxins were measured in the stomach contents of approximately half of the fish collected () and at only low levels, with maximum concentrations of 43 and 144 ng g−1, respectively, both measured in pinfish (L. rhomboides). In September and November 2005, brevetoxins were measured in all fish stomach contents. Surprisingly, the average concentrations in stomach contents in fish collected in September and November 2005 and May 2006 were not significantly different from each other (Kruskal–Wallis one-way analysis of variance by ranks followed by a Dunn's multiple comparisons test, 95% significance level). The highest level we measured in 2005 and 2006 was 2839 ng g−1 in the stomach of a scaled sardine (H. jaguana) collected in May 2006. However, the highest level reported in (3955 ng g−1) is from a spotted seatrout (C. nebulosus) collected in 2004.
3.4. Toxin profiles in fish
For both types of experiments (fish exposures to toxic clams and to K. brevis cultures), LC-MS analyses revealed that the fish had accumulated the same toxins to which they were exposed (). The toxin profile observed in the hard clams was almost identical to that observed in the liver of both pinfish and croakers, consisting principally of brevetoxin metabolites (cys-PbTx-2 and OxCys-PbTx-2) and, to a lesser extent, PbTx-3 (approximately 30% of the total amount of toxins). For planktivorous fish, the relative proportion of the toxins (PbTx-2>PbTx-3>PbTx-6>PbTx-9) observed in viscera was almost identical to that observed in the K. brevis culture (PbTx-2>PbTx-3,-9>PbTx-6>brevenal). Interestingly, brevenal, the brevetoxin antagonist produced by K. brevis was not detected in the viscera of these fish despite its presence in the K. brevis culture.
Brevetoxin composition in fish compared to their food sources in experimental exposures (K. brevis, toxic hard clams) and in selected fish species collected from St. Joseph Bay
Toxin profiles determined for fish collected live from St. Joseph Bay were mainly metabolized brevetoxins (). No brevetoxins could be identified by LC-MS in extracts from four additional fish (two pinfish, L. rhomboides; one scaled sardine, H. jaguana; and one Atlantic thread herring, O. oglinum). These sample extracts were less concentrated (150–850 ng ml−1 by ELISA) than those listed in for which profiles could be obtained. The lack of any detectable brevetoxins may be the result of insufficient sensitivity of our LC-MS method, the duration of sample storage prior to LC-MS analysis (6–30 months at −20 °C) or the presence of unidentified brevetoxin metabolites.