Download Re-evaluation of the Relationship between Pfiesteria and Estuarine

ECOSYSTEMS
Ecosystems (2003) 6: 1–10
DOI: 10.1007/s10021-002-0194-5
© 2003 Springer-Verlag
COMMENTARIES
Re-evaluation of the Relationship
between Pfiesteria and Estuarine
Fish Kills
Cavell Brownie,1* Howard B. Glasgow,2 JoAnn M. Burkholder,2
Robert Reed,2 and Yongqiang Tang1
1
Department of Statistics, North Carolina State University, Raleigh, North Carolina 27695, USA; and 2Center for Applied Aquatic
Ecology, North Carolina State University, Raleigh, North Carolina 27606, USA
INTRODUCTION
relationship. Based on his probability calculations,
Stow concluded that these results were “equivocal”
for implicating toxic Pfiesteria as a cause of fish kills
and recommended a surveillance program to improve understanding of the importance of toxic Pfiesteria as a fish kill stimulus.
In view of the important consequences of fish
kills to human populations and industries in the
mid-Atlantic region (Diaby 1996; Burkholder 1998;
Lipton 1998; MD DNR 1998), we believe that it is
necessary to reevaluate the relationship between
Pfiesteria and fish kills. Therefore, our intent here is
to show that if the calculations in Stow (1999) are
carried out for probability values that are consistent
with the existing data, then the criteria proposed by
Stow do in fact implicate toxic Pfiesteria as a causal
agent in fish kills. To justify this response, we note
that the type of surveillance that Stow recommended—sampling for toxic and potentially toxic life
stages of Pfiesteria spp. and sampling at an appropriate temporal scale (weekly to biweekly during seasons when toxic Pfiesteria outbreaks occur and
hourly or at 3- to 4-h intervals on sampling dates
when fish exhibiting signs of stress prior to death
are encountered)—is already in place and has been
ongoing for more than a decade (see, for example,
Burkholder and others 1995, 1997, 1999, 2001a;
Glasgow and others 1995, 2001a; Burkholder and
Glasgow 1997; Glasgow and Burkholder 2000). The
resulting database is readily accessible from the
North Carolina Department of Environment & Natural Resources (NC DENR 1998 –2001) and from
the laboratory of coauthors J.M.B. and H.B.G. (da-
In recent years, fish kills along the mid-Atlantic US
coast have become an increasing problem, with
important economic, environmental, and public
health implications (Glasgow and others 1995;
Burkholder 1998; Grattan and others 1998; Haselow and others 2001; Shoemaker and Hudnell
2001). Research into the causes of these fish kills is
ongoing, and monitoring and surveillance programs
have been instituted to investigate (among other
factors) the role of actively toxic forms of two
known species within the dinoflagellate genus Pfiesteria (Burkholder and others 1995, 2001a; Steidinger and others 1996; Burkholder and Glasgow
1997; Glasgow and others 2001b).
In their recent analyses of the relationship between Pfiesteria and fish kills, Burkholder and others
(1999) and Stow (1999) stated, as others have
noted previously (Meyer and Barclay 1990), that it
is difficult to establish the causes of estuarine fish
kills at the ecosystem level. The evaluation of Burkholder and others (1999) was based on the biology
and toxic behavior of Pfiesteria, as well as empirical
sampling of field fish kill events then in progress, as
supported by laboratory analyses of samples collected from each fish kill. In contrast, Stow (1999)
conducted theoretical probability calculations and
argued that information demonstrating the presence of toxic Pfiesteria during fish kills was insufficient to prove that there was a cause-and-effect
Received 17 September 2001; Accepted 11 April 2002.
*Corresponding author; e-mail: [email protected]
1
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C. Brownie and others
tabase; also in publications, for example Burkholder
and others 1995, 1997, 1999; Burkholder and Glasgow 1997; Glasgow and Burkholder 2000; Glasgow
and others 2001a). We used information provided
by the database to refine the calculations in Stow
(1999), leading to conclusions that differ from those
of Stow.
We used two approaches. First, the data from the
extensive monitoring program, including data from
fish kill events, were used to estimate probabilities
that were treated as unknown by Stow. Based on
these estimates, we then repeated the calculations
described in Stow (1999) for a realistic range of
conditions. Second, we used the data from fish kill
events in a retrospective, case-control type of analysis to test for an association between the presence
of actively toxic Pfiesteria and fish kills. Both sets of
results provided considerable evidence linking toxic
Pfiesteria and fish kills.
METHODS
Data Collection
Although toxic Pfiesteria-related fish kills have occurred in Chesapeake Bay (MD DNR 1998; Magnien and others 2000) as well as North Carolina
waters, we restricted our analysis to North Carolina
estuaries, consistent with Stow’s approach. Empirical sampling efforts have focused on the Neuse
Estuary, where most fish kills related to toxic Pfiesteria have occurred (Burkholder and Glasgow 1997;
Burkholder and others 2001a; Glasgow and others
2001a). In other North Carolina waters— for example, the Pamlico Estuary, Taylors Creek, and the
New River Estuary (Burkholder and others 1995,
1997; Burkholder and Glasgow 1997)—sampling
was conducted to characterize environmental conditions during certain fish kill events. The state
environmental agency (North Carolina Department
of Environment and Natural Resources [NC DENR],
located in Washington and Raleigh, North Carolina) provided additional background data. As
noted, the NC DENR fish kill database is computerized and easily accessible (NC DENR 1998 –2001);
in addition, various publications have summarized
information from it (for example, Burkholder and
others 1995, 1999, 2001a; Burkholder and Glasgow
1997; Glasgow and others 2001b).
In the Neuse Estuary, six stations on three
transects—Flanners Beach and Kennel Beach as
up-estuary transect stations, Beard Creek and Slocum Creek as mid-estuary stations, and Cherry
Point and Minnesott Beach as down-estuary
transect stations (Figure 1)— have been sampled
Figure 1. The Neuse Estuary monitoring sites. The map
shows the three major transects in the mesohaline Neuse
as the up-estuary transect (Flanners Beach [FLN] and
Kennel Beach [KELN] stations), the mid-estuary transect
(Beards Creek [BRD] station), and the down-estuary
transect (Minnesott Beach [MIN] and Cherry Point
[CHY] stations) located within the Albemarle/Pamlico
Estuary System of North Carolina, USA.
throughout the growing season (April through
Oct.), coinciding with the period when fish kills
generally occur, for a full suite of physical, chemical, and biological variables (including Pfiesteria).
Sampling was conducted weekly to biweekly in
1991–92 and weekly during 1993–2000 (Burkholder and others 1995; Burkholder and Glasgow
1997; Glasgow and Burkholder 2000; Glasgow and
others 2001a). During 1993–98, all stations were
additionally sampled biweekly during the winter
months (November through March) (for example,
see Glasgow and Burkholder 2000). The sampling
design for this biomonitoring project was thus based
on a geographic and temporal sampling scheme.
The geographic sampling strategy was implemented
to examine the spatial variability of study variables
within the Neuse Estuary. The temporal scheme
required repeated sampling at the same predetermined sites throughout the biomonitoring effort.
These geographic and temporal strategies were
linked to provide a statistical assessment of variation in the estuary during routine monitoring with
information from fish kills (for example, see Glasgow and Burkholder 2000) to evaluate toxic Pfiesteria as a possible causative factor of fish death
within the study area.
About 40 variables were analyzed for each date
(including depths profiles of temperature, salinity,
photosynthetically active radiation, pH, dissolved
oxygen, and redox; upper water-column samples
Pfiesteria and Estuarine Fish Kills
for fecal coliform bacteria and certain Vibrio spp.;
upper and lower water column samples for phytoplankton chlorophyll a, total biovolume, and total
cell number; the biovolume and cell number of taxa
comprising 5% or more of the total cells; Pfiesteria
stage abundance; suspended solids; and nutrients as
total nitrogen, ammonium, nitrate/nitrite, total
Kjeldahl nitrogen, total phosphorus, and soluble
reactive phosphate, with additional nutrient forms
added in the past 3 years). Samples were routinely
evaluated during the 1991–2000 monitoring effort
using water and/or sediments to see whether potentially toxic strains of Pfiesteria were present in the
study area. Although molecular probe techniques
to distinguish Pfiesteria from other lookalike species
in estuarine water samples were not available until
1998 (Rublee and others 1999; Oldach and others
2000; Bowers and others 2000), we applied these
techniques to archived samples to check/confirm
the presence/absence of toxic or potentially toxic
stages of Pfiesteria spp. For example, as illustrated by
Bowers and others (2000), water samples preserved
in acidic Lugol’s solution (Vollenweider and others
1974) are amenable to analysis by polymerase
chain reaction (PCR) methods. We used PCR to
recheck for Pfiesteria presence/absence in more than
500 selected water samples preserved in acidic
Lugol’s and held at 4°C in darkness, dating from
1991 to 1997. Thereafter, PCR analyses were completed on freshly collected samples. In response to
in-progress fish kills, water samples collected while
and where fish were dying were analyzed for approximately 40 physical, chemical, and biological
variables in an attempt to assess the cause(s) of fish
kills (Burkholder and others 2001c; Glasgow and
others 2001a).
Low-oxygen stress has often been associated with
fish kills in the Neuse Estuary (Burkholder and
Glasgow 1997; Burkholder and others 1999). Our
analyses included determination of dissolved oxygen concentrations both in the kill area and in
adjacent waters (from the outer edge of the kill
zone to a distance of approximately 1.0 km) that
could potentially have provided refuge habitats
(Burkholder and others 1999). Other factors such
as pesticide spills were detected infrequently by the
state environmental agency and were also considered in our interpretations (Burkholder and others
1995; Burkholder and Glasgow 1997; Glasgow and
others 2001a). We assayed for the presence of potential bacterial fish pathogens, including certain
Vibrio species such as Vibrio anguillarum and V.
vulnificus (Austin and Austin 1993). Although
harmful algae other than Pfiesteria spp. have not
previously been associated with fish kills in the
3
Neuse Estuary, we examined samples for the presence of potentially harmful species such as raphidophytes (Chattonella spp., Fibrocapsa japonica Toriumi & Takano, Heterosigma akashiwo (Hada)
Hada), potentially toxic and otherwise harmful
chrysophytes (for example, Chrysochromu lina, Phaeocystis), and other potentially toxic dinoflagellates
(for example, Karlodinium micrum [Leadbeater &
Dodge] J. Larsen, formerly Gyrodinium galatheanum;
Gymnodinium fuscum F. Stein, formerly Gyrodinium
aureolum; Karenia brevis (Davis) G. Hansen &
Moestrup, formerly Gymnodinium breve; which was
not expected in the mesohaline estuary but is
known from coastal North Carolina; Akashiwo sanguinea (Hirasaka) G. Hansen & Moestrup, formerly
Gymnodinium sanguineum) (see Daugbjerg and others 2000 for further taxonomic information).
We also conducted standardized fish bioassays
(Burkholder and others 1995, 1999, 2001c; Glasgow and others 1995, 2001a; Burkholder and Glasgow 1997; Glasgow and Burkholder 2000) to determine whether actively toxic Pfiesteria had been
present and so could have caused or contributed to
the kill event. The procedure follows Henle-Kochs’s
postulates (Evans 1976; Harden 1992), modified for
toxic rather than infectious agents, to determine
whether toxic Pfiesteria was involved in estuarine
fish kills. In early research, the data for Pfiesteria
fish-killing activity were cross-confirmed in parallel
work by the independent laboratory of Dr. E. Noga
(Noga and others 1993, 1996). The standardized
procedure has been cross-corroborated by Lewitus
and others (1995), and by Marshall and others
(2000). Standardized fish bioassays must be used in
fish kill evaluations for toxic Pfiesteria involvement
for the following reasons: First, light microscopy
cannot be used to distinguish Pfiesteria spp. from
numerous benign estuarine lookalike species (socalled “pfiesteria-like” organisms that physically resemble Pfiesteria), (Burkholder and Glasgow 1997;
PICWG 1999 –2002). Second, species-specific molecular probes—first available in 1998 for P. piscicida
(Rublee and others 1999) and in 1999 for P. shumwayae as Pfiesteria species ‘B’; (Oldach and others
2000)— can detect the presence of Pfiesteria spp.,
but they cannot discern whether they are in actively toxic (as opposed to nontoxic) mode (PICWG
1999 –2002; Burkholder and others 2001a; Rublee
and others 1999, 2001). Third, efforts to diagnose
whether actively toxic Pfiesteria spp. (or as yet undetected additional toxic pfiesteria-like species) are
involved in estuarine field fish kills or fish epizootics have remained handicapped because purified
Pfiesteria toxin is not yet available for developing
field-reliable assays for toxin detection (Fairey and
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C. Brownie and others
Figure 2. The surface of this three-dimensional graph
represents the values of P(fish kill 兩 toxic Pfiesteria) for all
values of P(toxic Pfiesteria) and P(fish kill) between 0.01
and 0.99 and with P(toxic Pfiesteria 兩 fish kill) ⫽ 0.38. The
lighter-shaded part of the surface represents values of
P(fish kill 兩 toxic Pfiesteria) for P(toxic Pfiesteria) and P(fish
kill) between 0.02 and 0.10, a range that includes the
respective estimates, 0.03 and 0.08, of these probabilities.
others 1999; Burkholder and others 2001a; KimmBrinson and others 2001; Melo and others 2001).
Therefore, properly conducted fish bioassays are
the only reliable technique now available to test for
the presence of actively toxic strains of Pfiesteria spp.
(and of other, as yet unknown toxic pfiesteria-like
dinoflagellates) from natural water or sediment
samples (Burkholder and Glasgow 1997; Marshall
and others 2000; Burkholder and others 2001a,c;
Samet and others 2001; see PICWG 1999 –2002 for
a consensus document defining much of the correct
terminology used in Pfiesteria research). The standardized fish bioassay procedure (Burkholder and
others 2001c; Samet and others 2002) is a powerful
tool in Pfiesteria-related fish kill assessment because
it provides a reliable, although conservative, means
to determine whether actively toxic Pfiesteria was
present at the estuarine kill while fish were dying.
Definitions and Estimates of Relevant
Probabilities
Stow (1999) attempted to evaluate the evidence for
toxic Pfiesteria as a cause of fish kills by comparing
the probability of a fish kill when toxic Pfiesteria was
present to the probability of a fish kill when toxic
Pfiesteria was not present. Stow stated that when the
ratio P(fish kill 兩 toxic Pfiesteria) : P(fish kill 兩 no toxic
Pfiesteria) is greater than 1, there would be evidence favoring Pfiesteria as a cause of fish kills. He
then calculated the value of this ratio for a wide
range of conditions and presented results in his
Figures 1 and 2 (compare to our Figures 2 and 3).
Figure 3. Values of the ratio P(fish kill 兩 toxic Pfiesteria)/
P(fish kill 兩 no toxic Pfiesteria) for P(toxic Pfiesteria) between 0.01 and 0.1, with separate curves for P(toxic Pfiesteria 兩 fish kill) ⫽ 0.28, 0.38, 0.52, and 0.66. The dashed
horizontal line corresponds to a value of 1 for the ratio
P(fish kill 兩 toxic Pfiesteria)/P(fish kill 兩 no toxic Pfiesteria).
However, that range of conditions was unrealistic
because it extended well beyond the narrow range
of plausible values for the chances of finding toxic
Pfiesteria, based on extensive field as well as laboratory data (Burkholder and Glasgow 1997; Burkholder and others 1999, 2001a). We repeated
Stow’s calculations for a more realistic, narrower
range of conditions. In our calculations, we have
used probability estimates based on the available
data from samples that were obtained in routine
monitoring and during fish kills.
As noted by Stow (1999), to obtain estimates of
the relevant probabilities, the probabilities must be
defined on consistent spatial and temporal scales.
Definitions must also be consistent with the objective of describing the role of toxic Pfiesteria in the
initiation of a fish kill. For the spatial dimension, we
considered the study region to be the approximately 103-km2 area covered by routine monitoring. Time was based on the 25 weeks of the year
(May through October) when more than 99% of
the fish kills occurred and monitoring was more
intensive. We considered fish kills over broad temporal and spatial scales (hours to weeks and 0.1 to
20 km2, respectively) (Burkholder and others 1995,
1999; Burkholder and Glasgow 1997; Glasgow and
others 1995, 2001a). The kill zone was defined to
extend from the area where most fish were dying
outward to the outermost edge of the area with
dying fish. Non–fish kill areas were sampled at a
distance of 70 m or more from the outer edge of the
Pfiesteria and Estuarine Fish Kills
kill zone (Burkholder and others 2001c). Fish kills
were assessed whenever dying fish, or dying fish
mixed with dead fish, were visible from a slowly
moving or anchored boat (8-m–long customized
Albemarle boat with central console) and involved
fish 3 cm or more in length.
The probability of a fish kill, or P(fish kill), was
defined as the probability that a fish kill was initiated (anywhere in the study region) during a random time unit. P(fish kill) was estimated by dividing
the number of time units during which a fish kill
was initiated (at any point in the study region) by
the total number of time units in the monitoring
period. For biological and operational reasons, we
took the time unit to be a day. Given 10 years of
monitoring (1991–2000) during a 25-week period
each year, the total number of time units was 10 ⫻
25 ⫻ 7 ⫽ 1750. The number of days when a fish kill
was initiated was 128, giving the estimate P(fish kill) ⫽
128/1750 ⫽ 0.07. The probability that actively toxic
Pfiesteria was present during a fish kill, or P(toxic Pfiesteria 兩 fish kill), was estimated by the proportion of
fish kill events during the monitoring period in which
toxic Pfiesteria was implicated as a causal agent. Given
that toxic Pfiesteria was implicated in 49 of a total of
128 fish kills, we estimated P(toxic Pfiesteria 兩 fish
kill) ⫽ 49/128 ⫽ 0.38. This estimate differs from the
value of 0.52 used by Stow (who used only data for
1991 to 1993) because the period that we considered
(1991–2000) included data from all years (1991–93,
1995–98) when fish kills related to toxic Pfiesteria
were reported (Burkholder and Glasgow 1997; NC
DENR 1998–2000; Burkholder and others 1999,
2001a; Glasgow and others 2001a).
Analogous to P(fish kill), we defined P(toxic Pfiesteria) as the chance that toxic Pfiesteria was present
in the study region on a randomly chosen day
during the monitoring period. Finally, P(fish kill 兩
toxic Pfiesteria) was the probability that a fish kill
was initiated in the study region given that toxic
Pfiesteria was present on that day. In his Eq. (3),
Stow (1999) expressed P(fish kill 兩 toxic Pfiesteria) in
terms of P(toxic Pfiesteria 兩 fish kill), P(fish kill), and
P(toxic Pfiesteria). He then evaluated P(fish kill 兩 toxic
Pfiesteria) for P(toxic Pfiesteria 兩 fish kill) ⫽ 0.05,
0.52, and 0.95, and for all values of P(fish kill) and
P(toxic Pfiesteria) between 0.01 and 0.99.
Rearranging Stow’s Eq. (3), we instead used estimates for P(fish kill) and P(toxic Pfiesteria 兩 fish kill)
to obtain plausible values for P(toxic Pfiesteria)
based on the following equation:
P共toxic Pfiesteria兲 ⫽ P共toxic Pfiesteria兩fish kill 兲
䡠 P共 fish kill 兲/P共 fish kill兩toxic Pfiesteria兲
To estimate the denominator P(fish kill 兩 toxic Pfiesteria), we noted that P(fish kill and toxic Pfiesteria) is
approximately equal to P(toxic Pfiesteria) because
laboratory toxicity assays were negative for toxic
Pfiesteria for all samples (more than 3000) collected
during routine monitoring (that is, not during a fish
kill event) (Burkholder and Glasgow 1997; Burkholder and others 1999, 2001c; Glasgow and others
2001a). Thus, P(fish kill兩 toxic Pfiesteria) ⫽ P(toxic
Pfiesteria and fish kill)/P(toxic Pfiesteria) ⬇ 1.0, and
P(toxic Pfiesteria) ⬇ 0.38 䡠 0.07 ⫽ 0.03.
Reconstructed Analysis with Data-based
Probabilities
Stow’s (1999) Figure 1 showed the values of P(fish kill
兩 toxic Pfiesteria) when P(toxic Pfiesteria 兩 fish kill) ⫽
0.05, 0.52, and 0.95, and for all values of P(fish kill)
and P(toxic Pfiesteria) between 0.01 and 0.99. We
produced a similar graph, although with P(toxic Pfiesteria 兩 fish kill) ⫽ 0.38. In that graph, we indicated the
small region that corresponds to realistic values of
P(toxic Pfiesteria) and P(fish kill) based on the estimates above—that is, P(toxic Pfiesteria) between 0.02
and 0.10, and P(fish kill) between 0.02 and 0.10.
In Stow’s (1999) Figure 2, the ratio P(fish kill 兩
toxic Pfiesteria)/P(fish kill 兩 no toxic Pfiesteria) was
graphed against P(toxic Pfiesteria) and P(fish kill) for
the same set of conditions used in his Figure 1. We
obtained a corresponding graph for a range of conditions that is consistent with the probability estimates obtained above. In addition, instead of a
three-dimensional graph, we obtained a two-dimensional graph by rewriting the ratio so that it is
a function of P(toxic Pfiesteria 兩 fish kill) and P(toxic
Pfiesteria) only:
P共 fish kill兩toxic Pfiesteria兲
P共toxic Pfiesteria兩fish kill 兲 䡠 P共 fish kill 兲 䡠 P共no toxic Pfiesteria兲
⫽
P共 fish kill兩no toxic Pfiesteria兲 P共toxic Pfiesteria兲 䡠 P共no toxic Pfiesteria兩fish kill 兲 䡠 P共 fish kill 兲
⫽
5
P共toxic Pfiesteria兩fish kill 兲 䡠 共1 ⫺ P共toxic Pfiesteria兲兲
共1 ⫺ P共toxic Pfiesteria兩fish kill 兲兲 䡠 P共toxic Pfiesteria兲
6
C. Brownie and others
Table 1. Summary of the Database of Major Fish Kills (Defined as Affecting at least 1000 Fish Following
Meyer and Barclay 1990)
Year or Period
Fish Bioassaysa (1991–2000, n ⬎2000 fish bioassays)
Pfiesteria-related fish mortality
Fish mortality related to other causes (handling
mortality, disease, pesticide spills, etc.)b
Fish Kill Eventsc (1991–2000)
Pfiesteria-related fish kills
Fish kills related to other causes
Number
Relationship(s)
ca. 1990
P (toxic Pfiesteria 兩 fish mortality) ⫽ 0.99
ca. 10
49
79
P (other causes 兩 fish mortality) ⫽ 0.01
P (toxic Pfiesteria 兩 fish kill) ⫽ 0.38
P (other causes 兩 fish kill) ⫽ 0.62
In fish bioassays, mortality is regarded as a proxy for estuarine fish death.
a
Compiled from Burkholder and others (1995, 1997, 1999); Burkholder and Glasgow (1997); and 1998 data from NC DENR (1998 –2000) see also Glasgow and Burkholder
(2000); Burkholder and others (2001a); Glasgow and others (2001a). All samples evaluated in fish bioassays were collected from in-progress fish kills.
b
These values represent fish death due to handling.
c
Data for 1991, 1992, 1993 are from Burkholder and others (1995). The 1992, 1993 data are from the fish kill records of the NC DENR Washington regional office, North
Carolina, and Raleigh, North Carolina; see NC DENR (1998 –2001). Data for 1995–98 are from Burkholder and Glasgow (1997); Burkholder and others (1997,1999); Glasgow
and Burkholder (2000); and NC DENR (1998 –2000). Data for toxic Pfiesteria estuarine fish kills in 1994 are not available because biohazard III facilities, needed to ensure
the health safety of laboratory staff when conducting fish bioassays with toxic Pfiesteria, were not available (Burkholder and Glasgow (1997); PICWG 1999 –2001; Burkholder
and others 2001c).
The ratio was calculated for values of P(toxic Pfiesteria) from 0.01 to 0.10 and for P(toxic Pfiesteria 兩
fish kill) ⫽ 0.28, 0.38, 0.52 and 0.66, wherein 0.38
is the best estimate based on currently available
data; 0.52 is the value used by Stow (1999); and
0.28 and 0.66 are 0.75⫻ and 1.75⫻ of 0.38, respectively. Resulting values of P(fish kill 兩 toxic Pfiesteria)/P(fish kill 兩 no toxic Pfiesteria) were graphed
against P(toxic Pfiesteria).
Case-Control Matched Pairs Retrospective
Analysis
A total of 128 major fish kills occurred in North
Carolina estuaries from 1991 through 2000 (NC
DENR 1998 –2000). For each fish kill, samples were
collected both within the area of the fish kill and in
a region immediately adjacent to the fish kill. We
summarized the presence or absence of toxic Pfiesteria in the paired regions (that is, fish kill and
non–fish kill areas) for the 128 fish kill events in a
2 ⫻ 2 table in the format commonly used for casecontrol matched pairs data. Here the cases were the
fish kills, the matched controls were the paired
non–fish kill areas, and toxic Pfiesteria was the risk
factor of interest (Breslow and Day 1980). McNemar’s test (with a continuity correction) was used
to test for an association between toxic Pfiesteria and
fish kills. The null hypothesis for this test can be
written as follows:
H 0:P共toxic Pfiesteria兩fish kill 兲
⫽ P共toxic Pfiesteria兩no fish kill 兲
Alternatively, the null hypothesis can be repre-
sented as equality of the odds of a fish kill when
toxic Pfiesteria was present and the odds of a fish kill
when toxic Pfiesteria was not present, or:
H 0:P共 fish kill兩toxic Pfiesteria兲/
P共 fish kill兩no toxic Pfiesteria兲 ⫽ 1
RESULTS
Presence of Toxic Pfiesteria.
Throughout the decade-long monitoring effort,
more than 3000 samples were collected from North
Carolina estuaries to determine whether toxic Pfiesteria is present under fish kill as well as non–fish
kill conditions. In evaluating these samples and others, the actively toxic form (functional type) of Pfiesteria has not been reported during non–fish kill
conditions (Burkholder and others 1995, 1999,
2001a,b; Burkholder and Glasgow 1997; Glasgow
and Burkholder 2000). However, in more than
2000 fish bioassays conducted with water samples
collected from areas where fish kills were in
progress, more than 99% caused fish mortality in
association with toxic Pfiesteria populations (Table
1). Those samples were from the 49 estuarine fish
kills that were linked to toxic Pfiesteria. The 49 kills
occurred within the area delineated by the upper
and lower transects (Figure 1), and about 90% of
them involved portions of one or more of the
transects (Burkholder and others 1995, 1999,
2001a; Burkholder and Glasgow 1997; Glasgow and
others 2001a). From 1991 to 2000, 49 of 128 fish
kills were related to toxic Pfiesteria in North Carolina
Pfiesteria and Estuarine Fish Kills
Table 2. Results from the Case-Control Matched
Pairs Retrospective Analysis
Non–Fish Kill Area
Fish Kill Area
Toxic Pfiesteria
No Toxic Pfiesteria
Total
7
itively, there would be evidence of an association
between the presence of toxic Pfiesteria and fish kill.
Case-Control Matched Pairs Retrospective
Analysis
Toxic
Pfiesteria
No Toxic
Pfiesteria
Total
0
0
0
49
79
128
49
79
128
waters, whereas 79 kills were attributed to other
causes (primarily, low dissolved oxygen) (Table 2).
Reconstructed Analysis
The surface in Figure 2 represents the values of
P(fish kill 兩 toxic Pfiesteria) for all values of P(toxic
Pfiesteria) and P(fish kill) between 0.01 and 0.99
and with P(toxic Pfiesteria 兩 fish kill) ⫽ 0.38. The
lighter shaded area in Figure 2 represents values of
P(fish kill 兩 toxic Pfiesteria) for P(toxic Pfiesteria) between 0.02 and 0.10 and P(fish kill) between 0.02
and 0.10 —ranges that include the respective estimates (0.03 and 0.07) of these probabilities. Comparison with the three graphs in Stow’s (1999)
Figure 1 shows that most of the values of P(fish kill
兩 toxic Pfiesteria) calculated by Stow (1999) are not
consistent with estimates of P(toxic Pfiesteria) and
P(fish kill) based on data from the intensive monitoring program and from fish kill events.
Figure 3 shows values of the ratio P(fish kill 兩 toxic
Pfiesteria)/P(fish kill 兩 no toxic Pfiesteria) calculated
for a range of conditions supported by the data,
with separate curves for P(toxic Pfiesteria 兩 fish kill) ⫽
0.28, 0.38, 0.52, 0.66, and P(toxic Pfiesteria) between 0.01 and 0.10. Comparing the curves with
the horizontal line corresponding to a value of 1 for
P(fish kill 兩 toxic Pfiesteria)/P(fish kill 兩 no toxic Pfiesteria), the value of the ratio is always more than 1
and usually substantially more than 1. Using Stow’s
(1999) argument that values for this ratio greater
than 1 support toxic Pfiesteria as a cause of fish kills,
Figure 3 provides evidence of such a causal relationship.
To interpret Figure 3, it may help to note that the
ratio P(fish kill 兩 toxic Pfiesteria)/P(fish kill 兩 no toxic
Pfiesteria) can be interpreted as the ratio of the odds
of finding toxic Pfiesteria when there is a fish kill to
the odds of finding toxic Pfiesteria on any day in the
study region. Thus, if toxic Pfiesteria was more likely
to be found in samples collected at the beginning of
a fish kill than in samples collected during routine
monitoring, the ratio would exceed 1 and, intu-
For the 128 recorded fish kills, actively toxic Pfiesteria was detected in the fish kill area but not in the
surrounding non–fish kill area in 49 cases, whereas
actively toxic Pfiesteria was absent in both fish kill
and the surrounding non–fish kill areas in 79 cases
(Table 2). There were no instances where actively
toxic Pfiesteria was detected in samples taken from a
non–fish kill area, regardless of whether or not
toxic Pfiesteria was detected in the associated fish kill
area. That is to say, toxic Pfiesteria was consistently
found only in fish kill areas during fish kills. The
corresponding (continuity corrected) Chi-square
value is 47.02 (P⬍0.0001), providing strong evidence that toxic Pfiesteria was positively associated
with the fish kills.
DISCUSSION
The main objective of Burkholder and others
(1995) was to describe fish kills linked to actively
toxic Pfiesteria; this paper reported data useful for
estimating P(toxic Pfiesteria 兩 fish kill). The paper
referred readers to other available sources (especially NC DENR 1998 –2000) for data from which
P(nontoxic Pfiesteria), P(toxic Pfiesteria) and P(fish
kill 兩 toxic Pfiesteria) could have been estimated.
Other publications (for example, Burkholder and
Glasgow 1997; Burkholder and others 1997,1999)
contained summaries of that information. Nevertheless, Stow based his calculations on Burkholder
and others (1995) alone and did not consider the
available data from many years of monitoring and
laboratory analyses (for example, Burkholder and
others 1997, 1999; Burkholder and Glasgow 1997;
NC DENR 1998 –2000).
Another issue addressed in Burkholder and others (1995, 1999) and Burkholder and Glasgow
(1997), but not considered by Stow (1999), is that
the evaluation of the presence/absence of actively
toxic Pfiesteria has consistently not relied on the
mere presence of Pfiesteria. Many so-called toxic
algae (as defined as in Burkholder 1999), including
Pfiesteria, are known to have populations that range
in toxicity status from benign (noninducible—incapable of killing fish with toxin in standardized fish
bioassays) to highly toxic (Gentien and Arzul 1990;
Anderson 1991; Skulberg and others 1993; Edvardsen and Paasche 1998; Bates and others 1998). The
mere presence of Pfiesteria does not ensure that the
8
C. Brownie and others
organism is responsible for fish kills because benign
and temporarily nontoxic as well as toxic functional
types of Pfiesteria can occur (Burkholder and Glasgow 1997; Burkholder and others 1995, 1999,
2001a, b). The important question concerning the
role of Pfiesteria in an estuarine fish kill is whether
actively toxic Pfiesteria was present at the inprogress kill. The diagnosis of toxic Pfiesteria involvement solely on the basis of the presence of the
organism has been discouraged (Burkholder and
others 1995, 1999, 2001a, c; Burkholder and Glasgow 1997; MD DNR 1998; Glasgow and Burkholder
2000; Magnien and others 2000; Glasgow and others 2001a), because none of the available techniques to detect Pfiesteria presence (for example,
Burkholder and Glasgow 1995; Steidinger and others 1996; Rublee and others 1999; Oldach and others 2000; Glasgow and others 2001b) can discern
whether it is in an actively toxic or a temporarily
benign mode (Burkholder and others 1995, 1997,
1999; Burkholder and Glasgow 1997; PICWG
1999 –2001; Burkholder and others 2001a, c). Instead, appropriately conducted fish bioassays are
required (Burkholder and others 1995, 1999,
2001a, c; Burkholder and Glasgow 1997; Lewitus
and others 1995; PICWG 1999 –2002; Glasgow and
Burkholder 2000; Marshall and others 2000; Glasgow and others 2001a).
The use of standardized fish bioassays (Burkholder and others 1995, 1999; Burkholder and
Glasgow 1997), a multistep procedure that follows
Henle-Kochs’s postulates modified for toxic rather
than infectious agents, permits evaluation of
whether a Pfiesteria population from an estuarine
water sample taken while and where fish were
dying was toxic when collected—that is, at the time
of the fish kill (Burkholder and others 2000c). As
Stow (1999) pointed out, there are no unambiguous a priori analytical methods of documenting a
toxic Pfiesteria-related fish kill within an estuarine
setting, nor is there a post mortem assay that can
forensically show toxic Pfiesteria as a cause of fish
mortality in an environmental setting. A field-reliable assay for Pfiesteria toxin, applicable for use in
water samples as well as fish tissue, may make it
possible to evaluate whether toxic Pfiesteria was involved in events that are detected and sampled after
fish kills (Burkholder and Glasgow 1997; Fairey and
others 1999; Kimm-Brinson and others 2001).
Given these limitations, however, Stow (1999)
failed to consider the importance of standardized
fish bioassays, which are the critical tool in determining toxic Pfiesteria involvement in estuarine fish
kills (PICWG 1999 –2001; Samet and others 2001).
Our calculations used the available data (1991–
2000) from both fish kills and monitoring. We recognize that values of the ratio in Figure 3 were
based on estimates rather than on known values of
probabilities. Also, the choice of time units used to
define the probabilities and to obtain estimates was
not without subjectivity. In generating Figure 3, we
therefore allowed for error in the estimates by using
a wide range of values for both P(toxic Pfiesteria 兩 fish
kill) and P(toxic Pfiesteria).
The results shown in Figure 3 and yielded by the
case-control analysis both demonstrate a positive
association between toxic Pfiesteria and fish kills.
That is, the analysis indicates that actively toxic
Pfiesteria essentially occurs only at fish kills. Even so,
as Stow cautioned, a strong positive association
cannot necessarily, on its own, be interpreted as
evidence of a cause-and-effect relationship. There is
still the possibility that an as yet unidentified factor
is causally associated with both fish kills and the
presence of toxic Pfiesteria. Within the past 2 years,
significant progress has been made in characterizing
a potent water-soluble toxin from actively toxic,
fish-killing Pfiesteria cultures (J. Ramsdell, P.
Moeller, personal communication; patenting process initiated) and characterizing its pharmacological activity (Kimm-Brinson and others 2001; Melo
and others 2001). Purified toxin will enable the
development of field-reliable toxin assays that will
provide an additional diagnostic tool to strengthen
insights about the extent to which toxic Pfiesteria
causes or contributes to disease and death in wild
fish.
A formal, detailed reevaluation of all available
peer-reviewed publications on Pfiesteria was recently published by a national science panel
charged with that task by the Centers for Disease
Control and Prevention (Samet and others 2001).
The panel endorsed findings by Burkholder and
others (1995, 1999) and Burkholder and Glasgow
(1997) indicating that there is compelling evidence
that actively toxic Pfiesteria can cause estuarine fish
kills, stating:
The preponderance of evidence from laboratory
and field investigations supports the proposition
that Pfiesteria has caused fish kills in estuaries of
Chesapeake Bay and the southeastern coastal regions of the United States. The evidence supporting the argument that blooms of the organism are
consequences— rather than causes— of fish kills
[as suggested by Stow 1999] is considerably less
persuasive. The behavior reported for Pfiesteria is
consistent with the considerable global experience with other ichthyotoxic algal bloom species,
Pfiesteria and Estuarine Fish Kills
their impacts, and established procedures applied
by harmful algal bloom researchers.
In contrast, the panel supported neither Stow’s
(1999) analysis nor his conclusions (Samet and others 2001):
Stow’s [1999] reservations are based primarily on
his misinterpretation that field sample data on
cell densities and presence of Pfiesteria were the
sole basis for inferring its active role in the observed fish kills. His largely statistical argument
ignores the laboratory-based experimental evidence that established the organism’s toxicity in
the first place (see Burkholder and Glasgow
1997), confirmatory fish bioassays, and the presence of toxic life stages during the fish kills (see
Tables 1 and 2 in Burkholder et al. 1995).
While Stow (1999) did recognize that Pfiesteria has
been lethal to fish in laboratory experiments, he did
not grasp the significance of the confirmatory laboratory bioassays for determining whether Pfiesteria
was actively toxic at in-progress kills.
Thus, extensive field and laboratory data, considered in this reevaluation, exist to support the
premise that actively toxic Pfiesteria is a causative
agent of major estuarine fish kills. Further insights
about the role of toxic Pfiesteria in estuarine fish kills
will require continuation of the current monitoring
programs, the application of a field-reliable assay
for Pfiesteria toxin, and ongoing intensive research
efforts to increase our understanding of the complex biological processes involved in this phenomenon.
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