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
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
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.