Summary of Maryland Pfiesteria Sampling and Results:
August 1, 1997 - October 22, 1997

Maryland Department of Natural Resources (10/22/97)

Documenting that Pfiesteria piscicida or a similar organism is the cause of fish health problems or a fish kill is a difficult and complex process. Samples must be collected during or immediately after a fish kill or lesion event, otherwise the Pfiesteria cells may revert to a non-toxic form. Positive identification of Pfiesteria in the sample can only be accomplished through scanning electron microscopy (SEM); a very laborious and time consuming procedure. Other techniques are available that provide a preliminary assessment; but these too require specialized equipment and training. There are currently only two laboratories capable of carrying out these analyses: Dr. JoAnn Burkholder's (North Carolina State University) and Dr. Karen Steidinger's (Florida Department of Environmental Protection).

Maryland DNR follows the four steps outlined below in its investigation to determine if Pfiesteria piscicida or a similar dinoflagellate is the cause of observed fish kills or lesion outbreaks;

STEP 1: Dead fish, a significant percentage of lesioned fish, or fish in distress are observed and no other explanation (such as low dissolved oxygen or a chemical spill) is apparent. At this point, many explanations for the event are possible.

STEP 2: Algal samples collected at the site of the event are examined under light microscope to determine if Pfiesteria-like cells are present and, if so, their density. Pfiesteria cannot be differentiated from many harmless dinoflagellates at this point. This step is a quick analysis to determine if a Pfiesteria-like organism is a possible explanation for the observed fish health problems and if proceeding to the next step is warranted. Results of this step are labeled as "preliminary". This step typically requires one to several days.

STEP 3: Bioassays (adding fish to live cultures of the dinoflagellate) are conducted to determine if the Pfiesteria-like organism is capable of killing fish or inducing lesions. If so, we can safely conclude that a Pfiesteria-like organism was active at toxic levels during the kill or outbreak and was at least partially responsible for the event. This step can currently only be conducted at Dr. Burkholder's laboratory. This step typically requires one to several weeks to complete.

STEP 4: SEM is carried out to identify the dinoflagellate species involved. Dr. Steidinger's laboratory cultures the samples with algal prey to promote growth of sufficient densities of dinoflagellate cells to then conduct SEM. This is only step which Dr. Steidinger's laboratory conducts (Dr. Burkholder's laboratory is capable of Steps 2, 3, and 4) and may require two months or more to complete.

Since August 1, 1997, Maryland DNR has collected 77 samples for Pfiesteria analysis. Most of these were collected on the Pocomoke (48), Kings Creek (6), and Chicamacomico (6) during fish kills or lesion outbreaks. The remainder were collected on Fishing Bay (1), Eastern Shore Wicomico Creek (1), Nanticoke (3), and Patuxent (9) rivers as part of DNR's rapid response to reported fish health problems. Most samples were sent to both laboratories, but practical limitations prevented Dr. Steidinger's laboratory from accepting samples from all sites.

To date (October 22, 1997) Maryland DNR has received at least preliminary results on 55 of the collected samples. The presence of Pfiesteria-like cells and positive bioassays (Step 3 above) has confirmed that Pfiesteria or a similar dinoflagellate was present and toxic on the Pocomoke River, Kings Creek, and Chicamacomico River at the time of the observed fish kills and lesion outbreaks. Light microscopy (Step 2 above) revealed the presence of Pfiesteria-like cells in some samples collected on the Wicomico Creek and Patuxent rivers, however there was no corroborating field evidence (i.e. fish kills, significant numbers of fish with Pfiesteria-like lesions, or fish in distress) to indicate a toxic outbreak of Pfiesteria. All other results were negative (no Pfiesteria or Pfiesteria-like cells present).

Preliminary results of SEM analysis (Step 4 above) have been received from Dr. Steidinger on four samples; three from the Pocomoke River and one from Kings Creek. These analyses reveal the presence of three unidentified Pfiesteria-like dinoflagellates. All three may be toxic and at least two have been found in association with fish kills elsewhere. Work is proceeding to further identify these three species. Although Pfiesteria has not been found in the four samples examined to date, laboratory results make it clear that a toxic, Pfiesteria-like species has been active on the three affected rivers. Dr. Burkholder has used SEM to confirm the presence of Pfiesteria cells in a sample from the Pocomoke River collected during May, 1997. Corroborating field evidence was not present at that time.

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