Bioassay for ichthyocidal activity of Pfiesteria piscicida: Characterization of a culture flask assay format

Abstract

The description of the heterotrophic dinoflagellate Pfiesteriapiscicida as the causative agent of fish lesions and deaths along the mid-Atlantic estuaries has revealed the need for bioassays to assess its potential toxigenicity. We designed a bioassay in which fish are exposed to clonal dinoflagellate strains or environmental consortia (e.g. environmental water or sediment samples) in 750-mL culture flasks, and examined the relationships among dinoflagellate proliferation profiles, the presence ofP. piscicida and fish deaths. Assay development and characterization were accomplished with dinoflagellate clonal cultures(P. piscicida, Karlodinium micrum,Prorocentrum minimum, CCMP1828, CCMP1829, and CCMP1834)co-incubated with sets of two young adult sheepshead minnows(Cyprinodon variegatus). Variables characterized included water quality (pH, dissolved oxygen, ammonia, nitrate and nitrite concentrations), the effect of the presence of fish on the proliferation and compositions of protist (dinoflagellate, protozoa, diatom) and bacterial populations, and time of fish death. The presence of fish in experimental flasks induced proliferation of P. piscicida and Cryptoperidiniopsis sp. (but not K.micrum or P. minimum) with populations rising between days 6 and 10, and declining 4 to 5 days later. Some fish deaths occurred when or soon after P. piscicida cell numbers were maximal. We conclude that this assay enables the assessment of acute effects of ichthyocidal dinoflagellates on fish during the first 10 days (StageA) of the experimental course. Fish deaths during the subsequent 10 to 20 days(Stage B) may be attributed to the proliferation of ichthyocidaldinoflagellates, pathogenic bacteria and/or deteriorating water quality,where as those beyond a period of approximately three weeks (Stage C) can be most certainly attributed to deteriorated water quality. Application of the flask assay to environmental samples [n=53] yielded fish deaths in all three stages:A, 78%; B, 11%, and C, 2%, with fish still living in 9% of the sample waters tested at the conclusion of the experiment beyond three weeks. The majority of samples that resulted in fish death in stage A tested positive for P.piscicida by PCR. If implemented with cautious interpretation, this assay should prove useful in monitoring blooms for the presence of P.piscicida and other dinoflagellate s pecies potentially harmful to fish.