Hemolysis, Fish Mortality, and LC‐ESI‐MS of Cultured Crude and Fractionated Golden Alga (Prymnesium parvum)¹

Abstract

Erythrocyte lysis and fish mortality assays, in combination with high performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis, were investigated for bioassay-guided fractionation of cultured golden alga (Prymnesium parvum). Intracellular constituents from isolated cell pellets and extracellular supernatant growth medium were fractionated by a variety of common separation modes, including reversed phase and normal phase solid phase extraction step fractionation procedures. For reversed phase fractionation of extracellular growth medium, one fraction was obtained that displayed hemolytic activity and adversely affected fish survival. Effective dose concentrations for this sample were similar in both assays and the LC-ESI-MS analysis of the fraction showed a number of mass spectral signals which were distinct to this fraction. Fractions obtained from separation of an ethanol extract of the lyophilized cell pellet provided one sample that was highly hemolytic, but not toxic to fish. Discrepancies such as this, along with notable fish behavioral responses from other nonhemolytic cell pellet fractions, problems with the use of unbonded silica gel for fractionation, and misleading mass spectral signatures are interesting in the context of our current understanding of P. parvum toxicity and remain to be investigated further. This work provides an account of ongoing research aimed toward comprehensive elucidation of toxic constituents produced by golden alga for the purpose of providing a better understanding and means to potentially remediate the ecological impact of this harmful bloom organism.(KEY TERMS: erythrocyte lysis; fish mortality; liquid chromatography; electrospray ionization; mass spectrometry; prymnesin.)

Effect of different salinities on growth and intra- and extracellular toxicity of four strains of the haptophyte Prymnesium parvum

“…In contrast, extracellular toxicity in P. parvum measured with a bioassay varies less, e.g. with EC 50 = 10 × 10 3 to 100 × 10 3 cells ml −1 (Rhodomonas test; Freitag et al 2011, present study), ED 50 = 100 × 10 3 to 500 × 10 3 ml −1 (fish test; Baker et al 2007, Schug et al 2010) and EC 50 = 2.5 × 10 3 to 350 × 10 3 cells ml −1 (Artemia test; Edvardsen & Paasche 1992).…”

“…Large variations in toxicity, intracellular (Uronen et al 2005, Hagström 2006) as well as extracellular (Skingel et al 2010), have been reported during the different phases of Prymnesium parvum growth. Extracellular toxicity can show a maximum during late exponential growth (Skingel et al 2010), but no particular growth phase was identified in our study.…”

“…bacteria) into the surrounding water. Bacterial abundance was not measured during the course of the present experiment, and more sampling points during exponential stage would be informative to confirm the stability of the measured C:N:P ratios, as for instance the C:P ratio of P. parvum cells has been found to vary between 50 and 150 during exponential growth (Skingel et al 2010). …”

“…The chemical substances enabling mechanisms (1), (2), partly (3) and toxicity to fish are insufficiently described; still, they are generally attributed to a col lection of compounds known as prymnesins (Manning & LaClaire 2010). However, recent work (Henrikson et al 2010, Schug et al 2010 suggested that P. par vum toxins responsible for organismal toxicity may not be the classic prymnesins described by Igarashi et al (1996). It is not yet clear if the toxins stored intracellularly, causing mortality of P. parvum grazers due to ingestion (mechanism 2) (Tillmann 2003, Barreiro et al 2005), are similar to the toxins released extracellularly, which may function as competitive mechanisms or aid in immobilization of prey cells for mixotrophic nutrition (mechanisms 1 and 3) (Skovgaard & Hansen 2003, Tillmann 2003.…”

“…As hemolytic assays are often not sensitive enough to determine P. parvum exotoxin production (Uronen et al 2005), bioassays with organisms such as the cryptophyte Rhodomonas salina (Fistarol et al 2003, Tillmann 2003, the brine shrimp Artemia (Meldahl et al 1994) or fish (Reich & Parnas 1962, Schug et al 2010) are used as an alternative. Since intra-and extracellular toxicity are not necessarily linked, both need to be measured to evaluate P. parvum toxicity (Parnas 1963, Manning & La Claire 2010.…”