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]

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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

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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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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 ⫽

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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

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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

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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

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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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