Development of an indirect quantitation method to assess ichthyotoxic b-type prymnesins from prymnesium parvum

Harmful algal blooms of Prymnesium parvum have recurrently been associated with the killing of fish. The causative ichthyotoxic agents of this haptophyte are believed to be prymnesins, a group of supersized ladder-frame polyether compounds currently divided into three types. Here, the development of a quantitative method to assess the molar sum of prymnesins in water samples and in algal biomass is reported. The method is based on the derivatization of the primary amine group and subsequent fluorescence detection using external calibrants. The presence of prymnesins in the underivatized sample should be confirmed by liquid chromatography mass spectrometry. The method is currently only partly applicable to water samples due to the low amounts that are present. The growth and cellular toxin content of two B-type producing strains were monitored in batch cultures eventually limited by an elevated pH. The cellular toxin contents varied by a factor of ~2.5 throughout the growth cycle, with the highest amounts found in the exponential growth phase and the lowest in the stationary growth/death phases. The strain K-0081 contained ~5 times more toxin than K-0374. Further investigations showed that the majority of prymnesins were associated with the biomass (89% ± 7%). This study provides the basis for further investigations into the toxicity and production of prymnesins

HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL−1, and the limit of detection was found to be 0.03 μg·mL−1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source

The blue mussel Mytilus edulis is vulnerable to the toxic dinoflagellate Karlodinium armiger-Adult filtration is inhibited and several life stages killed.

Blooms of the toxic dinoflagellates Karlodinium armiger and K. veneficum are frequently observed in Alfacs Bay, Spain, causing mass mortality to wild and farmed mussels. An isolate of K. armiger from Alfacs Bay was grown in the laboratory and exposed to adults, embryos and trochophore larvae of the blue mussel, Mytilus edulis. Adult mussels rejected to filter K. armiger at cell concentrations >1.5·103 cells ml-1. Exposure of adult mussels (23-33 mm shell length) to a range of K. armiger cell concentrations led to mussel mortality with LC50 values of 9.4·103 and 6.1·103 cells ml-1 after 24 and 48 h exposure to ~3.6·104 K. armiger cells ml-1, respectively. Karlodinium armiger also affected mussel embryos and trochophore larvae and feeding by K. armiger on both embryos and larvae was observed under the microscope. Embryos exposed to low K. armiger cell concentrations suffered no measurable mortality. However, at higher K. armiger cell concentrations the mortality of the embryos increased significantly with cell concentration and reached 97% at 1.8·103 K. armiger cells ml-1 after 29 h of exposure. Natural K. armiger blooms may not only have serious direct effects on benthic communities, but may also affect the recruitment of mussels in affected areas

Mortality of <i>Mytuilus edulis</i> embryos and trochophore larvae after exposure to <i>Karlodinium armiger</i> at four different concentrations.

<p>Mortalities of embryos and trochophore larvae, separately and when pooled as a function of <i>K</i>. <i>armiger</i> cell concentrations. Data presented as mean values with SE bars.</p

<i>Karlodinium armiger</i> tube feeding on <i>Mytilus edulis</i> embryos and trochophore larvae.

<p>Initial tube feeding by <i>K</i>. <i>armiger</i> on a mussel embryo (A). <i>Karlodinium armiger</i> cells attached to a mussel embryo causing the vitelline coat to disrupt and release of egg content (B). <i>Karlodinium armiger</i> attracted to egg content released from a disrupted embryos (C). <i>Karlodinium armiger</i> tube feeding on a mussel trochophore larva (D).</p

Mortality of <i>Mytilus edulis</i> exposed to six <i>Karlodinium armiger</i> concentrations for 24 and 48 h.

<p>LC₅₀ values of 9.4·10³ ± 2.7·10³ cells ml^-1 and 6.1·10³ ± 0.3 ·10³ cells ml^-1 were found after 24 and 48 h, respectively. Data presented as mean values with SE bars.</p

Clearance rate of <i>Mytilus edulis</i> exposed to <i>Karlodinium armiger</i> and <i>Rhodomonas salina</i>.

<p>The mussels were exposed to a low and high bio-volume equivalent concentration of each algal species. Data presented as mean values with SE bars.</p