1999 Overview Summary of Pfiesteria and Fish Health monitoring in Chesapeake Bay


Prepared by:

The Maryland Pfiesteria Study Team

March 8, 2000


Introduction

A coordinated monitoring effort developed in response to fish and human health events in 1997-98 and continued with significant advances during 1999. Programs included water quality and habitat analyses, field surveys of fish health, field experiments on fish health in the Pocomoke River, pathology and histology of fish, and watershed pollution assessment. Advances in molecular techniques to the level of field application were incorporated into the monitoring and rapid response efforts. The latest techniques allowed, for the first time, for Pfiesteria monitoring to be closely integrated with monitoring of fish health and water quality on a Baywide scale.

Results

Advances in fish husbandry with menhaden have pushed the science toward improved experimentation. Baseline parameter estimates of fish health for menhaden were developed for better evaluations of fish condition in the future. Ulcers on fish were studied intensively and the relationships that may exist between ulcers on fish, water quality and the presence of Pfiesteria were examined. Due to the discovery of a myxosporidean parasite this year associated with post larval and early juvenile menhaden that were outwardly healthy or exhibited anomolies, there are additional insights and questions on the development of ulcers in fish connected with their habitat.

Characterization of systems with and without Pfiesteria continues to support the observation of relatively higher nutrient and phytoplankton concentrations in areas where Pfiesteria is present in the water column. Work conducted by the team examining watershed characteristics has demonstrated that alternative fertilizer applications on systems with phosphorus enriched soils can reduce nutrient loading to the Bay.

No fish kills in 1999 were attributable to toxic Pfiesteria events. Pfiesteria piscicida was, however, associated with a severe and prolonged fish health event involving ulcerated menhaden on the Middle River and found at the site of a human health event on Back Creek that fit the current definition for "Possible Estuary Associated Syndrome".

Overall menhaden catches were greater in 1999 versus 1998 but percent of menhaden with ulcers has continued to decline since 1997 (1997: 9.1%, 1998: 2.1%, 1999: 1.7%). Geographically, lesion prevalence in 1999 was clustered in tributaries at the head of the Bay (e.g., Middle River) and the lower Eastern Shore. Assessment of anomaly rate in the broader fish community sampled across fisheries programs indicates a low bay-wide lesion incidence, approximately 0.5%, similar to 1998. Background prevalence has been reported as 0.5% in Mid-Atlantic and 0.7% in Gulf cost estuaries. In general, Chesapeake Bay fish appear to be healthy although there may be localized or species specific problems.

Pathology reports from several cooperating agencies concluded that both ulcerated and superficially normal mehaden from the lower Eastern Shore tributaries in May and June had severe myxosporidian infections, later identified as Kudoa sp. In addition to the Kudoa spores, a highly invasive, plasmodial stage suggestive of a myxozoan was found in the tissues of the Pocomoke menhaden during late May through early July. Some of the invasive stage lesions were associated with ulcers and chronic inflammatory infiltrates. Bacterial and viral assays on a sample of ulcerated and normal fish were negative, indicating that the invasive sporozoan was most likely the primary pathogen causing these small ulcers.

Summaries of results for the program areas are attached and give more detail to the highlights of the work for 1999.


Table of Contents Page

Section 1. Associations between Pfiesteria, Fish health and Environmental Conditions in

Chesapeake Bay....................................................................................................... 3

Section 2. Fish Health Aspects of 1999 Pfiesteria Investigations in Maryland....................... 10

Section 3. Comprehensive Fish Health (Lesion) Sampling - 1999.......................................... 19

Section 4. Pocomoke Watershed Pollution Assessment........................................................... 24

Summary: Associations between Pfiesteria, Fish health and Environmental Conditions in Chesapeake Bay.


Prepared by:


Robert Magnien, Dave Goshorn, Bruce Michael, Peter Tango and Renee Karrh

Tidewater Ecosystem Assessment

Maryland Department of Natural Resources

580 Taylor Avenue

Annapolis, MD 21401


Draft-March 8, 2000


Introduction

In the summer of 1997, the Pocomoke River, Kings Creek on the Manokin River, and the Chicamacomico River, all tributaries of the Chesapeake Bay, experienced toxic outbreaks of Pfiesteria piscicida . The evidence in support of this conclusion included the combination of the following factors: identification of P. piscicida from the water column by scanning electron microscopy; enumeration of a sufficient density of Pfiesteria-like cells in the water column at the times of the events to account for toxicity; fish kills with no other apparent cause after an evaluation of numerous factors; and confirmation of Pfiesteria toxicity in water samples assayed using fish. Atlantic menhaden (Brevoortia tyrannus) in the outbreak areas suffered mortalities, high percentages of lesions, and incidences of distressed behavior. Medical studies revealed significant cognitive deficits in humans exposed to the affected water bodies (Grattan et al. 1998).

These outbreaks led to the implementation of a comprehensive monitoring and research effort by the State of Maryland and its partners to evaluate the factors that are associated with outbreaks of P. piscicida and related species with potential toxicity. This monitoring effort has included a rapid response capability to evaluate sites where fish health, human health, or other conditions suggest that a toxic outbreak of Pfiesteria may be present.

Objectives

The objectives for the 1999 sampling, analysis and interpretation were the following:

Rapid Response

1. Provide for the rapid response testing of, and reporting on, water quality, habitat, Pfiesteria-like organisms and associated biological communities in cases of suspected toxic outbreaks.

2. Provide water quality, habitat and Pfiesteria data needed to fulfill DNR's obligations under Maryland's "Protocol for Closing and Reopening Rivers Affected by Pfiesteria or Pfiesteria-like Organisms."

Environmental Assessments

3. Increase understanding of critical habitat factors contributing to the frequency of toxic outbreaks of Pfiesteria-like organisms.

4. Evaluate relationships between critical habitat factors and human activities.

5. Identify areas vulnerable to outbreaks of toxic Pfiesteria or Pfiesteria-like organisms.

6. Identify the principal harmful algal bloom species and their distribution in Maryland's waters, including support for development of molecular probes.

7. Track improvements in water bodies affected by Pfiesteria-like organisms that have implemented management programs.

8. Provide rigorous exposure data to epidemiologists studying the relationship between toxic Pfiesteria-like dinoflagellates and human health.

Methods

In response to the 1997 outbreaks, six Chesapeake Bay tributary systems and two tributary systems on the Atlantic Coastal Bays were chosen for intensive sampling starting in 1998. These tributaries are in the region affected in 1997 and include five systems that share similar water quality and watershed characteristics with the three that had outbreaks. Sampling for most elements of the monitoring program is conducted once to twice monthly between April and October.

Water quality samples are taken along the longitudinal axes of the tributary systems and include temperature, salinity, dissolved oxygen, and a broad suite of nutrient (N, P, C, Si) fractions.

Phytoplankton sampling is conducted using several techniques. Chlorophyll samples for biomass are collected at all water quality stations, species composition are collected at a subset of these stations, and continuous in vivo fluorescence measureents of chlorophyll are made from longitudinal cruises in all of the tributary systems.

Water column sampling for Pfiesteria and related dinoflagellates in the water column was conducted initially only in areas that had potential outbreaks. Taxonomic identification has been conducted by Dr. JoAnn Burkholder (North Carolina State University) and Dr. Karen Steidinger (Florida Department of Environmental Protection). This process has been conducted via reading of plates on the armored dinoflagellate from photographs of samples prepared under scanning electron microscopy. Bioassays to assess toxicity to fish have been conducted by Dr. Burkholder's laboratory. Starting in 1998, and to a greater extent in 1999, sampling was conducted routinely at a subset of water quality stations in the affected region as well as in a number of tributaries around the Chesapeake Bay. The recent development of a polymerase chain reaction (PCR)-based genetic probe for P. piscicida and two related dinoflagellates by Dr. David Oldach of University of Maryland Institute for Human Virology, Dr. Parke Rublee, University of North Carolina, Greensboro and Dr. Gerardo Vasta of the University of Maryland Center of Marine Biotechnology, has greatly enhanced our ability to detect these species in a matter of hours from the time of collection. During potential outbreaks, samples are analyzed by the PCR probe and by light microscopy to ascertain whether or not there are "Pfiesteria-like" cells and, if so, their density. If the samples are positive for Pfiesteria or Pfiesteria-like cells, subsamples are incubated in two cultures, one with fish and one with algal prey to determine potential toxicity.

Sediment sampling for Pfiesteria and related dinoflagellates was conducted in 1998 and 1999 at approximately 300 stations in 14 tributaries throughout the Chesapeake and Coastal Bays to determine Bay-wide distributions. Stations for sediment sampling were chosen randomly within a geographic area that included the 3-12 ppt summer salinity zone where Pfiesteria has been typically found in Chesapeake Bay and at a depth of less than 2 m. All sediment samples were incubated in two cultures, one with fish and one with algal prey.


Results

Water Quality Relationships with Pfiesteria

Overall water quality in the region affected in 1997 was assessed using the new monitoring data. In general, most nutrient (N, P, C) fractions, especially the dissolved organic fraction, were found to be elevated when compared with a broader array of stations sampled throughout the Chesapeake Bay and its tributaries. Most stations in the affected Lower Eastern Shore region were above the 60th percentile for total N and P pools and above the 80th percentile for dissolved organic N and P pools. Reaches of tributary systems that had outbreaks in 1997 and have also tested positive for Pfiesteria in the water column in subsequent years appear to have among the highest nutrient concentrations in this region.

Using 1999 data, water quality was compared in areas with and without P. piscicida as determined by PCR probe. From the set of stations where P. piscicida was found in the water column (i.e. positive), a geographic area that encompassed the salinity range of 3-12.5 ppt (95% confidence interval on the mean salinity for positive samples) and the time frame of July - October was established as the most likely space - time dimension for Pfiesteria to be present in the water column. Water quality data from stations in this same space - time dimension, but where Pfiesteria was not found (i.e. negative), was used for comparison with the positive set. This "negative" set was then further divided by eliminating tributaries was found active in 1997 or 1998. This comparison showed that almost all total, organic and particulate nutrient pools were higher in waters where Pfiesteria was active in the water column; chlorophyll was also higher.

Elevated chlorophyll has been associated with Pfiesteria in both North Carolina and Maryland. In the 1997 outbreaks in the Pocomoke R., longitudinal profiles showed that the chlorophyll maximum was in the vicinity of the fish kills during August, the month of the outbreaks (MD DNR 1998). A working hypothesis developed based upon data collected in the Pocomoke R. suggests that high chlorophyll levels may serve as a rich food supply both for the heterotrophic P. piscicida and for the menhaden that are associated with toxic outbreaks. The detection of the longitudinal position of chlorophyll maxima has, therefore, been of particular interest and continuous flow-through in vivo fluorometry (IVF) has been employed to improve its detection.

In 1999 P. piscicida was detected at numerous stations and dates in the Transquaking R. and to a lesser extent in the Chicamacomico R. The 1999 summer-averaged (July-October) chlorophyll for the Chicamacomico/Transquaking system was highest in the Transquaking where summer averages were above 40 ug/l.

During most of the sampling period in 1999 on the Pocomoke River, the chlorophyll maximum region included Shelltown, the vicinity of the 1997 outbreaks. During the period, chlorophyll levels remained below 40 ug/l. In 1999 only one of many water column samples (n=73) were positive for the presence of P. piscicida.

As seen in past years, the chlorophyll for the Manokin/Kings Cr./Back Cr. system in 1999 sustained algal blooms of over 80 ug/l in its smaller upper tidal reaches during summer. In 1999 P. piscicida was detected near a report of a human health problem in Back Cr. during August. In 1997, a Pfiesteria outbreak occurred in Kings Cr.


Pfiesteria piscicida Distribution in Chesapeake Bay

Pfiesteria piscicida appears to be widespread throughout tidal tributaries of Chesapeake Bay. An initial investigation in 1992 revealed the presence of P. piscicida in Jenkins Creek, a tributary of the Choptank River. Since 1997, a variety of methods have been utilized to test for the presence and toxicity of P. piscicida. The occurrence of four separate fish health events, all involving kills, lesion outbreaks, and erratic fish behavior on the Pocomoke, Manokin, and Chicamacomico rivers in August and September 1997, prompted the analysis of water samples from these rivers. Scanning electron microscopy (SEM) and fish bioassays confirmed the presence of toxic P. piscicida at the time and place of all four events. Recently developed PCR methodologies from two independent laboratories confirmed the presence of P. piscicida during lesion outbreaks on the Wicomico and Chicamacomico rivers in 1998 and the Middle, Gunpowder and Bohemia rivers in 1999. PCR also confirmed the presence of P. piscicida on Back Creek (a tributary of the Manokin River) in response to a reported incident of possible human exposure to P. piscicida toxin in 1999. Bioassays conducted on water from all the 1998 and 1999 events did not detect the toxic form of the dinoflagellate. During 1999, a survey approach was employed in which water samples were collected monthly (April - October) from approximately 50 sites in Chesapeake Bay tributaries and subjected to PCR analysis. This survey revealed P. piscicda on the Pocomoke, Wicomico, Chicamacomico/Transquaking system, Potomac, Magothy, Patapsco, and Back rivers. One sample collected in 1998 on the Rhode River and subjected to PCR analysis also revealed P. piscicida. Finally, approximately 300 sediment samples have been collected on 14 Chesapeake and Coastal bay tributaries during 1998 and 1999. Approximately 15% of these samples have been analyzed to date with P. piscicida being detected via PCR and SEM in sediment from the Chicamacomico River.

Pfiesteria shumwayae has been detected in seven Maryland tributaries. Analysis of sediment samples have revealed its presence in the Big Annemessex, Manokin, Wicomico, and Chicacacomico rivers in the Chesapeake Bay drainage, and Trappe Creek in the Coastal bays. Polymerase chain reaction analysis has also revealed P. shumwayae in the water column of the Potomac and Middle rivers.


Summary

Information collected between 1997 and 1999 as part of Maryland's monitoring of environmental conditions related to Pfiesteria has significantly increased our understanding of this organism and its relationships with environmental conditions. P. piscicida is widespread in Maryland, having been found in 14 tributary systems of the Chesapeake Bay, with the greatest occurrences, and the only obvious toxic outbreaks, on the mid-Delmarva Peninsula. Given the broad distribution of tributaries in which it has been confirmed present thus far, it is likely that further sampling will reveal its presence in many other tributaries. Pfiesteria piscicida is not, however, necessarily common in these tributaries. In many cases, P. piscicida's presence has been confirmed by one or a few of many samples collected over multiple months or years. On the other hand, there are several tributaries that have consistently demonstrated P. piscicida activity. The Chicamacomico, in particular, has demonstrated significant P. piscicida activity annually since 1997. The Manokin and, to a lesser extent the Pocomoke and Wicomico, have also demonstrated P. piscicida activity on multiple years. Evidence to date suggests that this activity is concentrated in the July through October period. To date, only the four events in 1997 have strong evidence on P. piscicida present in a toxic state.

The recently named Pfiesteria shumwayae has also been confirmed in Maryland tributaries. Evidence to date has confirmed its presence in seven tributaries. The data for this species is far less extensive than that for P. piscicida, however preliminary evidence suggests that it's distribution in the water column is not as widespread as P. piscicida. To date, there has been no evidence of P. shumwayae toxicity in Maryland waters, however, P. shumwayae collected in Maryland sediment samples has repeatedly demonstrated ichthyotoxicity in laboratory settings. Laboratory evidence suggests that P. shumwayae's life history and toxic capabilities are similar to P. piscicida (J.M. Burkholder, personal communication). Polymerase chain reaction probes are now available for P. shumwayae, allowing future work to better describe its distribution.

The 1997 through 1999 data indicate several associations between Pfiesteria, fish health and environmental conditions in Chesapeake Bay. In all incidences (n=7) where young-of-the-year Atlantic menhaden were found with high percentages of lesions for extended periods of time, P. piscicida was also found in the water column, and there is strong evidence that it was in a toxic form at four of these events. While there is strong evidence that toxic P. piscicida was responsible for the fish kills and erratic behavior observed in the 1997 events, the relationship between the dinoflagellate and menhaden ulcers is less clear. The field evidence presented here does not prove a mechanistic relationship between P. piscicida and the menhaden ulcers observed, but rather demonstrates a strong association. There have also been cases demonstrated in 1999 of Pfiesteria presence in the water column without associated fish health problems. Several hypotheses have been proposed (some suggesting a mechanistic relationship) to explain the observed association between Pfiesteria and fish lesions ("Special Report of the Technical Advisory Committee on Harmful Algal Outbreaks in Maryland: Causes and Significance of Menhaden Lesions", February 12, 1999) and are being pursued as part of Maryland's response. This association between menhaden health and Pfiesteria, regardless of the underlying relationships, confirms the value of utilizing fish health as one of several indicators of possible Pfiesteria activity.

Areas where Pfiesteria has been found consistently in the water column have relatively high nutrient and chlorophyll levels; dissolved organic nutrients are particularly high. The positioning of the chlorophyll maxima in affected tributaries often coincides with where Pfiesteria is detected and is being closely evaluated as an indicator of outbreak occurrence. Nutrient enrichment continues to be a common denominator in systems that have exhibited the greatest and toxic Pfiesteria activity. A large part of the State's response continues to focus on understanding the complex interactions of specific habitat parameters that contribute to Pfiesteria presence and toxicity.


Acknowledgements

We wish to acknowledge the many contributors to the extensive coordinated efforts of this project that include work by Sally Bowen and the Maryland Department of Natural Resources Field Operations Center, Harry Rickabaugh, Rudy Lukakovic and Harley Speir of MD DNR Fisheries Service, Dr. Cindy Driscoll, Dr. Steve Jordan, Brett Coakley and Brenda Kibler of the Sarbanes Cooperative Oxford Lab, Charlie Poukish and the Maryland Department of the Environment Fish Kill Response Unit, Dr. JoAnn Burkholder and Dr. Howard Glasgow of North Carolina State University, Dr. David Oldach and Holly Ann Bower at University of Maryland, Dr. Park Rublee of University of North Carolina State University in Greensboro. Water quality analysis has been conducted by the Chesapeake Biological Laboratory and Department of Health and Mental Hygiene. We appreciate the many hours of in-house work contributed by Jeffrey Kiess, Margaret McGinty, Lee Karrh, Thomas Adams, Tony Allred, Calvin Jordan, Katheleen Freeman, Beth Ebersole, Bill Romano, Lenora Dennis, Renee Randall and Heather Soulen of the Maryland Department of Natural Resources, Tidewater Ecosystem Assessment Division in their assistance with data preparation, data analysis and/or preparation of this report. We thank Richard Lacouture and the Academy of Natural Sciences, Estuarine Research Center for their contributions in in vivo fluorescence work and plankton dynamics assessments.


References

Grattan, L. et al. 1998. Learning and memory difficulties after environmental exposure to waterways containing toxin-producing Pfiesteria or Pfiesteria-like dinoflagellates. The Lancet 532-539.




Summary: Fish Health Aspects of 1999 Pfiesteria Investigations in Maryland. Draft Summary Report, February 24, 2000



Prepared by:



Brett Coakley, Brenda Kibler, Cindy Driscoll, Stephen J. Jordan

Maryland Department of Natural Resources, Sarbanes Cooperative Oxford Laboratory

904 S. Morris St.

Oxford, Maryland 21654



John M. Jacobs, Reginal M. Harrell

University System of Maryland, Center for Environmental Sciences

Horn Point Laboratory

P.O. Box 775

Cambridge, Maryland 21613



Ana Baya

Maryland Department of Agriculture, Animal Health Laboratory

8077 Greenmead Drive

College Park, Maryland 20740



Renate Reimschuessel

Food and Drug Administration, Center for Veterinary Research

8401 Muirkirk Road

Laurel, Maryland 20708



James D. Hedrick, Lara B. Ras, F. J. Margraf

Maryland Cooperative Fish and Wildlife Research Unit

University of Maryland Eastern Shore

1120 Trigg Hall

Princess Anne, Maryland 21853

Introduction

Beginning in the fall of 1996, and continuing in the spring and summer of 1997, commercial and recreational fishers in the Pocomoke River and Pocomoke Sound region reported unusual numbers of fish with severe lesions and other abnormalities. Several species of estuarine and freshwater fish were involved. A variety of possible causes for the abnormalities was suggested, including unusual water quality conditions (low salinity and pH, especially in the spring of 1997), toxic discharges from agricultural, municipal or industrial sources, epizootic fish diseases, and toxic algal blooms. The 1997 events stimulated increased attention to fish health questions in Maryland, in part because of the need to protect the public from exposure to potentially toxic waters, and in part because there were important unanswered questions about the interactions of toxic Pfiesteria-like organisms with estuarine fish. A coordinated group of studies was initiated in the summer of 1997 to attempt to answer the many questions surrounding the fish health problems.

Intensive fish health investigations were undertaken in the Chesapeake Bay tidal tributaries in 1997 and 1998. These studies included: 1) fishery-independent sampling using bottom trawls, beach seines and cast-nets; 2) fishery-dependent observations conducted by biologically-trained observers on commercial fishing vessels; and 3) experimental studies designed to document the effects of confining fish under conditions reflecting both the estuarine environment and the potential stresses of commercial fishing gear (i.e. pound nets and bank traps). Tissue samples were collected from several species of fish for histopathological and microbiological examination during these studies.

The 1997 fish health study results from the Pocomoke River and Sound indicated an unusually high percentage of physical abnormalities, especially external lesions. Baywide studies, with fishery-independent sampling for lesion incidence in several tributaries , were conducted in 1998 and 1999. (Rickabaugh et al. 2000). The central findings of their work were 1) a Baywide prevalence of fish lesions (all species combined) of 0.54% in 1998 and 0.46% in 1999, and 2) lower prevalence of lesions on menhaden in 1998 (2.1%) and 1999 (1.7%) than in 1997 (9.1%).

Atlantic menhaden with external ulcers exhibited two general pathologies; both types were chronic and active, but distinguished by the presence or absence of granulomatous tissue (Jordan and May 1998, Jordan et al. 1999). Chronic active lesions are those that have been present for some time (days) based upon the appearance of well-developed immune responses, and that show no signs of healing. Granulomas reflect a particular type of immune response, and have been closely associated with ulcerative mycosis in menhaden and other species (Sindermann 1988), although granulomas can be induced by a variety of pathogens. The pathology results provided evidence that more than one factor was involved in the high prevalence of fish lesions in the Pocomoke River during 1997. A variety of opportunistic bacterial species was cultured from internal tissues of affected fish, with no consistent evidence of epizootic bacterial disease in the populations. Fungal hyphae were observed in histological sections of menhaden from each of the four 1997 morbidity and mortality events linked to P. piscicida, including Pocomoke Sound, Kings Creek and the Chicamacomico River. In 1998, menhaden with severe ulcerative lesions were collected from many of the same tributaries, as well as the Wicomico River (Wicomico Co). Unlike 1997, in 1998 there were no reported mortalities associated with any of these lesion events. Histological analyses of samples from these tributaries yielded results similar to those in 1997; the lesions were diagnosed by pathologists as mycotic granulomatosis (Jordan et al.1999).

There is an important question about how or whether these ulcers are spatially and temporally connected with toxic Pfiesteria-like blooms. Although P. piscicida toxin can cause epidermal necrosis under experimental conditions, its role in formation of chronic ulcers has not been determined (Lewitus et al. 1995, Burkholder and Glasgow 1997a, Noga 1998). Pathological and microbiological investigations have indicated frequent occurrence of fungal infections in the ulcers of menhaden (Sindermann 1988, Kane et al. 1998). Since the ulcers are chronic (days to weeks old), they are poor indicators of immediate toxic conditions at a particular time and place.

The origins of ulcers in fish are often difficult to discern, because most of our information is obtained during episodic events, or after the lesion has been infected by multiple secondary invaders. In most instances, disease in fish reflects an imbalance in the relationships of the host, pathogen, and environment. May and Sindermann (1999) proposed that ulcers originate from one of three major events: 1) direct physical injury to epithelium by fishing gear, predators, or other causes, 2) underlying systemic infections such as virus, fungi, or bacteria, or 3) stress-induced cellular or tissue changes resulting in necrosis. It is apparent in the literature that resolving the question of lesion origin requires directed experimental studies and detailed histological and physiological examination of infected fish. Since 1997, the Sarbanes Cooperative Oxford Laboratory (SCOL) has been working with researchers at the University of Maryland, U.S. Geological Survey, and Food and Drug Administration to determine the nature and progression of fish lesions in Chesapeake Bay. This report summarizes our collective 1999 findings in the areas of monitoring, histology, physiology, and experimental studies.


Objectives of 1999 studies

1. Document the prevalence and nature of fish abnormalities in commercial fisheries of the lower Pocomoke River and Pocomoke Sound.

2. Investigate the origins, nature and progression of skin abnormalities in Chesapeake Bay fish populations where the involvement of toxic P. piscicida is indicated.

3. Establish experimental conditions to re-create the skin abnormalities in a controlled mesocosm system.

4. Document the pathology and microbiology of fish afflicted with skin abnormalities.

5. Integrate the data with data from other components of the study through GIS and database management.


Methods

Onboard Commercial Fisheries Observation Program - Fisheries observations were conducted from both pound nets and bank traps in the Pocomoke River and Sound in 1999. The initial onboard observations began in April of 1999; regularly scheduled events started in July and continued through September of 1999. Fish from the commercial catch were identified to species and examined for specific skin anomalies. A total of 10,218 fish was sampled.

Menhaden Collection and Husbandry - Collection of larval menhaden was conducted by the University of Maryland Eastern Shore United States Fish and Wildlife Service Cooperative Unit (USFWS Coop. Unit) starting in late February, 1999. A modified ichthyoplankton tow fitted with a collection jar was used to capture fish at various locations. Late larval stage menhaden were brought to the Horn Point Aquaculture Facility (HPL) from April 23, 1999 through May 20, 1999 and immediately started on a brine shrimp diet (Artemia franciscana). The majority of fish were held in 500 gallon flow-through tanks fed by ambient unfiltered Choptank River water (6-15 ppt throughout the summer), with one batch (~500) placed in a fertilized 0.5 acre pond. Through metamorphosis, fish were fed a combination of Artemia, T. isochrysis, and a commercially available salmon starter diet onto which they were gradually weaned. Approximately 1000 additional juveniles were collected in July with cast nets on the Choptank River and mantained at HPL.

Physiology, Histopathology and Microbiology - Blood chemistry, hematology, acetylcholinesterase, histology and microbiology samples were collected from adult and juvenile menhaden from May through November 1999 during both lesion and non-lesion events. To establish normal ranges for blood variables and acetylcholinesterase, 95 adults were sampled from pound nets on the Pocomoke and Choptank Rivers. Eighty healthy juveniles were collected from the Choptank River. Variables examined include hematocrit, differential white cell counts, brain acetylcholinesterase levels, and serum protein, osmolarity, glucose, and chlorides. Additional blood chemistry, hematology, and acetylcholinesterase samples were collected in August 1999 in response to three outbreaks of lesions in the Wicomico, Manokin, and Middle River systems. The same variables were examined from 30-100 fish per location.

Histopathological examinations were conducted on 292 YOY Atlantic menhaden from a variety of sites and sampling programs during 1999. If available, five to ten (depending on size) YOY menhaden were fixed for histological analysis from each tributary per sampling date. The Fisheries Service began sampling juvenile menhaden in the Spring of 1999 earlier than previous years, since our goal was to document the beginning of the ulcerative lesions, and follow their progression throughout the summer. Histology slides were prepared at SCOL following standard methods and examined by Dr. Renate Reimschuessel, FDA and other cooperating pathologists.

Cage Studies - At each of the three lesion outbreaks described above, cage studies were conducted to determine whether healthy fish exposed to the ambient environmental conditions would develop the same type of ulcerative lesions. White perch (Morone americana) maintained at the DNR Vienna facility were transported to Back and Shiles Creeks on 8/24/99. Ten fish were placed in each of four 2'x2'x4' wire mesh cages (2 per site). Cages were retrieved after 48 hours and 96 hours with all fish examined for external damage, blood hematology and chemistry, and neurology. The details of cage design, deployment, and transport are described in Jacobs et al. 1999.

Atlantic menhaden (90-110mm) reared in the HPL hatchery were transported on 9/8/99 to the Middle River in fish live shipping boxes at a density of 15 per box. Three cages were deployed in Hopkins Creek and Norman Creek. Cages were pulled at 48, 96, and 144 hours. An additional cage was added to each site on 9/12/99 and allowed to remain for 312 hours. Two cages were deployed on 9/11/99 in the Choptank River off of the HPL pier to serve as controls and were pulled at 96 and 288 hrs. Fish were examined for external abnormalities, with 6 fish bled for hematocrit and protein determination, and preserved for histology. An additional 2 - 6 fish per cage were frozen immediately for Ache determination. Rapid response water quality and pfiesteria detection samples were taken from each system during each sampling day.


Results

Onboard Commercial Fisheries Observation Program - Of the 10,218 fish sampled, 0.352% (36 individuals) were observed with some form of skin anomaly and 0.078% (8 individuals) of the observed fish possessed a severe skin anomaly (erosions or ulcers). The 1999 percentage of fish with anomalies was comparable to the 1998 percentage of 0.306% (Hedrick et al. 1998). The percentage of severe skin anomalies, however, was lower than 1998 (0.192%). Atlantic menhaden showed a very low percentage of overall anomalies (0.30%; 8 fish), and most of these (6 fish) were affected by external parasites. Abrasions were the most commonly observed skin anomaly, followed by parasites, erosions and ulcers. No correlation between fish size (total length) and the occurrence of skin anomalies was observed for any species.

Menhaden Collection and Husbandry - Collection and rearing of larval, pre-juvenile, and juvenile stages of Atlantic menhaden was successful in 1999. In all, over 6,000 fish were raised to juvenile stages. However, initial mortality often exceeded 50% from time of collection to tank acclimation. Growth of menhaden equaled that noted in the wild until late September when growth rate declined, most likely due to reduced feeding and crowding in tanks. Transportation in "fish bags" (plastic bags filled half with water and half with pure oxygen) or a circular tank were both successful, with minimal mortality. The raised areas found in juveniles on the Pocomoke (possibly associated with Kudoa sp.), were not noticed in hatchery reared fish, although screening efforts were not undertaken. However, by mid-August, approximately 50% of the individuals in a single tank developed ulcers stemming from the maxillary area of the left side of the mouth. We believe the ulcers were initiated by rubbing on the sides of tanks, as the condition always was observed on the tank-wall side of the fish. The condition also was noted in fish held at the Virginia Institute of Marine Science (VIMS). While it is unlikely that the ulcers are of any overall health concern, VIMS researchers are examining the ulcers histologically. Hatchery juveniles were used for experimental work by researchers at HPL, the United States Geological Survey, VIMS, and the University of Maryland, Aquatic Pathobiology Center.

Physiology, Histopathology and Microbiology - Expected ranges for blood variables and acetylcholinesterase were determined from healthy adult and juvenile menhaden collected throughout 1999. Multivariate analysis of juvenile and adult data indicated significant differences between the life stages(p<0.01). Further examination of individual least square means suggested that the overall difference was driven by lower chlorides and glucose in juveniles and higher protein levels. The establishment of normal blood chemistry, hematology, and acetylcholinesterase ranges in menhaden had not previously been attempted, and gives us a valuable reference for future sampling. Although these values cannot be assumed to be true baseline levels, they represent a range encountered with normal sampling techniques for healthy fish.

In May, the Fish and Wildlife Health Program (FWHP) documented small raised areas on juvenile menhaden in the Pocomoke River (Somerset Co.). Samples were taken over the next few weeks for pathology and microbiology. By June, the prevalence of the raised lesions decreased and small ulcerative lesions were documented more frequently. The overall percentage of sampled fish with ulcerative lesions reached its peak of 18% on June 3rd. The lesion prevalence then began to decline; no fish were collected with significant exterior lesions after July 8th.

Pathology reports from several cooperating agencies concluded that both the ulcerated and superficially normal fish had severe myxosporidian infections, later identified as Kudoa sp. In addition to the Kudoa spores, a highly invasive, plasmodial stage suggestive of a myxozoan was found in the tissues of the Pocomoke menhaden during late May through early July. These parasites penetrated and surrounded muscle bundles, causing grossly observable raised lesions in 70% of the cases. In a number of fish, these parasites were also found in the visceral organs, branchial arches and intraocular muscles. Some of the invasive stage lesions were associated with ulcers and chronic inflammatory infiltrates. Bacterial and viral assays on a sample of ulcerated and normal fish were negative, indicating that the invasive sporozoan was most likely the primary pathogen causing these small ulcers.

Ulcerated menhaden were captured during routine sampling by DNR biologists on Shiles Creek, Wicomico River in August 1999. Blood chemistry and acetylcholinesterase sampling revealed no differences from Choptank River controls (p>0.05) however, no ulcerated fish were collected. Interestingly, slightly elevated neutrophil and monocyte counts (above control levels) were detected, indicating a moderate immune response.

In August, 1999 an advisory was issued for portions of Back Creek, Manokin River, due to three individuals who reported skin irritations and flu-like symptoms following water contact. Over 90 juvenile menhaden were sampled with no ulcerative lesions noted. However, the fish mouth isopod Olencira praegustator was noted in most fish, and caused mechanical opercular sores in 18% of the fish. In many cases, gill damage was extensive regardless of opercular sores. Hematocrit and white cell differentials in these fish were well within normal ranges. However, slight depressions of glucose, protein and chloride concentration were noted, along with elevation of serum osmolality. These changes could reflect gill damage caused by the parasite, altering osmotic capabilities and influencing feeding. Acetylcholinesterase levels were similar to control (normal) values (p>0.05).

On August 22nd, 1999 ulcerated menhaden were discovered in abundance in the Middle River, near Essex, MD. The lesion incidence varied from day to day but affected rates were as high as 88% (Rickabaugh et al. 2000). No other species of fish, including other clupeids, was affected with ulcerative lesions. Blood chemistry analysis of ulcerated fish showed signs of hypoglycemia, depressed protein, and red cell anemia as compared to normal values and other response locations. These conditions are consistent with hemodilution, or thinning of the blood through water entry, as well as infectious disease in the case of protein. Interestingly, the ulcerated Middle River fish were maintaining osmotic balance and chloride levels despite large open sores. It is difficult to explain the exact mechanism by which this was accomplished without a detailed examination of other electrolytes. The salinity in the Middle River (7-8ppt, 200-232 mmol/kg) at the time of collection was fairly close to "normal" internal salt concentrations (9-12 ppt, 260-348 mmol/kg) in menhaden. It is also known that muscular exertion releases lactate ions which can be reflected in total osmolality through gradient creation causing a net water movement into blood cells (Wood, 1991). As expected, differential white cell counts suggested an elevated cellular immune response with high neutrophil and monocyte counts. Acetylcholinesterase values were again similar to control values. Of great interest was the response of externally healthy fish collected at the Middle River. Again, elevated neutrophil and monocyte counts were noted, as well as high hematocrit, hyperglycemia, and mild hypochloremia. Although sampling techniques remained constant at all locations, it was possible that sampling influenced the general stress response noted in these fish. However, elevated differential counts suggested the stress may have been chronic. Clearly, as juvenile menhaden are schooling fish and theoretically all subjected to the same event or events preceding lesion development, more attention needs to be directed at those without lesions.

After repeated pathological and microbiological sampling, it was concluded that the lesions from Middle River menhaden were very similar to the lesions seen in both 1997 and 1998. The ulcers were heavily infected with fungi as well as several bacterial organisms. However, there were no common bacterial isolates taken from these fish; they were infected with a variety of gram-negative bacteria, so the bacterial infections were believed to be secondary. The pathological diagnosis for fish caught from the Middle River was essentially the same as the ulcerated fish collected from the Wicomico and Chicamacomico in 1998: chronic mycosis. Granulomatous tissue was seen in most of the fish examined, indicating that the infections had been progressing for some time. The descriptions of the lesions varied somewhat; some lesions were more red in color and characterized as being "fresh" and others were more necrotic and gray or white in color. The latter were characterized by some as "old" lesions. There was granulomatous tissue in both types of lesions, indicating that the "fresh" and "old" categories were both describing chronic lesions, and should not be used for field descriptions. Pathologists from other cooperating agencies concluded similar diagnoses.

Cage Studies - Cage studies conducted with white perch at Back Creek, Manokin River and Shiles Creek, Wicomico River revealed no abnormalities after 96 hours of exposure. This information, in combination with physiological assessments of juvenile menhaden collected, suggested no adverse conditions at these locations.

Extensive cage studies were conducted in Hopkins and Norman Creeks on the Middle River with juvenile Atlantic menhaden. Overall, mortality was 20% and occurred within 48 hours, most likely reflecting transport and acclimation stress. No lesions attributable to pathogens were noted, with no significant differences found between sites, times, or cages for hematocrit, protein, or acetylcholinesterase (p >0.05). Presumptive cell counts and probe results indicated that Pfiesteria-like cells were at low levels during the caging experiment.

Overall, the incidence of external anomalies on commercially harvested fish in the Pocomoke River and Sound in 1999 was similar to 1998, and much lower than in 1997. While the trend of sporadic, small scale lesion events continued, the Middle River episode was the first time these abnormalities were reported from a heavily populated Western Shore area. As in 1997 and 1998, histology results from ulcerated juveniles showed a high degree of bacterial colonization and granulomatous tissue, suggesting that the initiating event or events occurred well prior to their discovery. Unsurprisingly, menhaden with ulcerative lesions showed many signs of physiological stress, mostly stemming from hemodilution. It is apparent from this event response sampling that more attention should be paid to healthy fish found with ulcerated individuals; as schooling fish should all be subjected to the same event or events which preceded lesion development. The lack of changes in actetylcholinesterase at all sample sites may reflect lack of toxic organisms. However, we have not had the opportunity to test our hypothesis of inhibition under conditions where Pfiesteria toxin is present. Perhaps the most significant finding is the discovery of another invasive pathogen, tentatively described as of the genus Kudoa. Year 2000 sampling should aid in answering many of these questions through progression studies with those showing clinical signs of Kudoa, and continued examination of healthy and ulcerated Atlantic menhaden.


References

Burkholder, J.M. and H.B. Glasgow, Jr. 1997a. Trophic controls on stage transformations of a toxic ambush-predator dinoflagellate. J. Euk. Microbiol. 44(3):200-205.

Hedrick, J. D., L. B. Ras, and F. J. Margraf. 1998. Distribution, progression, and species specific incidence of fish skin abnormalities in the Pocomoke River system. 1998 3rd Quarterly Report. Maryland Department of Natural Resources, Cooperative Oxford Laboratory. Oxford, Maryland.

Kane, A.S., D.Oldach and R. Reimschuessel. 1998. Fish lesions in the Chesapeake Bay: Pfiesteria-like dinoflagellates and other etiologies. Maryland Med. J. 47(3):106-112.

Jacobs, J.M and R.M. Harrell. 1998. Causes, progression and probable outcomes of fish skin abnormalities in the Pocomoke River system. Univ. of Maryland, Horn Point Laboratory. Summary report, Dec. 4, 1998.

Jordan, S.J. and E.M. May. 1998. Histological and microbiological findings on fish taken from the Pocomoke River and adjacent tributaries. Maryland Dept. of Natural Resources, Cooperative Oxford Laboratory. Draft Report, Feb. 13, 1998.

Jordan, S.J., B.E. Coakley, B.J. Kibler, C.P. Driscoll, J. D. Hedrick, L.B. Ras, F.J. Margraf, J.J. Jacobs, R.M. Harrell, A.M. Baya, J. Evans. 1999. 1999 Fish health aspects of Pfiesteria invesigations in Maryland. Maryland Dept. of Natural Resources, Cooperative Oxford Laboratory. Final Report, Feb. 3, 1999.

Lewitus, A.J., R.V. Jesien, T.M. Kana, J.M. Burkholder, H.B. Glasgow Jr., and E.May. 1995. Discovery of the "phantom" dinoflagellate in Chesapeake Bay. Estuaries 18:373-378.

Noga, E. J., J. F. Levine, M. J. Dykstra, and J. H. Hawkins. 1988. Pathology of ulcerative mycosis in Atlantic menhaden Brevoortia tyrannus. Diseases of Aquatic Organisms 4:189-197.

Noga, E.J., L. Khoo, J.B. Stevens, Z. Fan and J.M. Burkholder. 1996. Novel toxic dinoflagellate causes epidemic disease in estuarine fish. Mar. Pollution Bull. 32(2):219 - 224.

Rickabaugh, H., R. Lukacovic and H. Speir. 1999. Comprehensive Fish Health Sampling. Draft report, Maryland Dept. of Natural Resources, Annapolis.

Sinderman, C.J. 1998. External ulcers of fish: some general considerations. Ulcerative Lesions in Fish: Causes and Effects. 26p. Easton Maryland, August 11-13, 1998.

Sinderman, C.J. 1999. External ulcers of fish: some general considerations. In Causes and Effects of Ulcerative Lesions in Fish (Edited by Jordan, S.J., Sinderman, C.J., Rosenfield, A., and May, E.B.) pp. 18 - 27. Maryland Department of Natural Resources, Oxford, MD.

Wood, C.M. 1991. Acid-base and ion balance, metabolism, and their interactions after exhaustive exercise in fish. J. Exp. Biol. 160:285-308.






Summary: Comprehensive Fish Health (Lesion) Sampling - 1999



Prepared By:



Harry Rickabaugh, Keith Whiteford and Harley Speir

Maryland Department of Natural Resources

Fisheries Service

Matapeake Work Station

301 Marine Academy Drive

Stevensville MD, 21666



Introduction

The appearance of fish with various types of ulcers has been linked to activity by Pfiesteria piscicida, a toxic dinoflagellate (Noga et al. 1996). Pfiesteria's toxin is thought to erode the slime coating (and possibly the epithelia), allowing the entry of fungi and bacteria which causes an ulcer. Maryland has adopted a surveillance program that uses ulcerated fish as a bio-indicator of Pfiesteria and other harmful algal blooms (HAB's). A program was established by the DNR Fisheries Service in 1997 (Speir and Pyle 1998) to sample fish from water bodies statewide to detect ulcers as an indicator of HAB's. Atlantic menhaden, Brevoortia tyrannus, are thought to be a species particularly vulnerable to the toxin and subsequent ulcer formation. Monitoring of areas closed due to presumed Pfiesteria toxin caused fish kills was begun as a directed activity in 1997. In 1998, staff were added and sampling was initiated in areas thought to be at risk of Pfiesteria activity (Lukacovic et. al. 1999). The 1998 sampling effort was repeated in 1999 with some modification.

Objectives

1. Provide information on the incidence and distribution of fish lesions in Chesapeake Bay and Maryland's Oceanside tributaries.

2. Serve as a platform for researchers to provide healthy and diseased fish for histopathological and microbiological analysis.

3. Respond to citizen calls into the DNR Fish Health Hotline.


Methods

Monitoring in 1999 of fish health and habitat quality was conducted on four levels. Level I was termed Rapid Response deployments. A 24 hour Fish Health Hotline was maintained by the Natural Resources Communication Center in the Tawes Office Building. This Hotline received calls from citizens regarding fish health problems. Fisheries Service biologists staffed the Rapid Response Team and responded to potential problems identified through the Hotline. An agreement between Maryland Department of the Environment Fish Kill Unit and Fisheries Service assured a timely response to fish kill or fish health reports.

For Level I investigations, fish communities were sampled with beach seine or cast net. Water samples for Pfiesteria and nutrients along with physical parameters were taken when dead fish were present, or more than 20% of one fish species with ulcerative lesions were collected in a sample of greater than 50 fish.

Level II sampling targeted those rivers which had been closed in 1997 (Pocomoke River, King's Creek and Chicamacomico River). These systems were sampled on a bi-weekly basis.

Level III sampling focused on water bodies with water quality and watershed characteristics similar to Level II rivers and therefore thought vulnerable to development of harmful algal blooms. Sample sites were located on Fishing Bay, Newport Bay, and the Nanticoke, Wicomico, Annemessex, Manokin, and St. Martin rivers and sites were sampled on a bi-weekly basis.

Sampling methods for Levels II and III were identical and sampling days often collected data from both Level II and III sites. Surface trawls had a 5 ft by 5 ft opening with the net body of 3/8 in stretch mesh. The codend was lined with 500 plankton netting. Sites were at specific river mile designations. Exact locations were selected based on site logistics and freedom from snags. One 6-minute trawl was made at each site parallel to the channel. Seine hauls, using a 100 foot net with 1/4 inch mesh, were made at the nearest snag-free beach. Tributaries of each major river were sampled at access points with 20 casts of a six foot cast net at each site. Sampling began in April and ended in October.

Catch was enumerated and twenty fish of each species measured. Any abnormalities such as lesions, mechanical damage or parasites were noted. For reporting purposes, anomalies were considered to be all types of external abnormalities with the exception of parasites. Ulcers on menhaden were characterized as new, old or recovering. All individuals with abnormalities were measured. At the request of fish health specialists, fish samples were retained for histology, microbiology or other analysis. Only Atlantic menhaden were retained from surface trawls. Water samples for analysis of Pfiesteria or nutrients were taken on request. Water quality (dissolved oxygen (D.O.), temperature, pH, conductivity and salinity) was recorded at each sampling site. The distribution of Atlantic menhaden captured during level II and level III monitoring was analyzed with the water quality measurements of temperature, D.O., and salinity. Each of these parameters were equally divided into predetermined ranges for this analysis.

For Level IV sampling, all Fisheries Service projects were asked to note the frequency and types of anomalies on fish that were collected in the various fishery dependant and fishery independent samples taken in Maryland tidewater. Projects included an angler survey on head boats out of Crisfield, the Striped Bass Juvenile Seine Survey, Striped Bass Tagging Survey, Crab Trawl Survey, Bay Pound Net Survey, Coastal Bays Trawl and Seine Survey, Baseline Fish Health Survey and Biological Indicators Trawl Survey.


Results

A total of 347,081 resident and migratory finfish were sampled by 8 surveys from 34 water bodies in Maryland's portion of the Chesapeake Bay, and ocean-side tributaries, to monitor for the presence of anomalies or lesions believed to be Pfiesteria-related. In 1997 and 1998 188,824 and 372,675 fish were sampled respectively. System-specific incidence of anomalies ranged from 0.0 to 29.8%. Lesion prevalence among all fish species sampled by these programs in 1999 was 0.46%.

Atlantic menhaden was the fish of most concern because of associations found between ulcerated menhaden and Pfiesteria. A total of 83,800 menhaden were collected in 1999 representing 24.1% of all fish sampled. In 1998, 48,789 menhaden were collected accounting for 12.6% of all fish sampled, and 16,622 menhaden were collected in 1997 accounting for 8.8% of the total catch. Lesion prevalence among menhaden was lower in 1999 (1.7%) than in 1998 (2.1%) or 1997 (9.1%) in all systems that were sampled. This trend was also seen in systems which were intensively monitored because of closures in 1997: Chicamacomico River, Kings Creek and Pocomoke River. In 1997, 22.3% of 5,754 menhaden had lesions; 11.2% of 3,012 had them in 1998; and 0.7% of 38,547 had lesions in 1999. Geographically, lesion prevalence in 1999 was clustered in the Head-of-Bay and lower Eastern Shore.

The prevalence of lesions among menhaden collected by cast net from the Chicamacomico River in September through November 1997 (n=865) and 1998 (n= 1783) was 69.1 and 15.6% respectively compared to 0.6% in September through November 1999 (n=2729). Lesion frequency also declined during the same time period in the other two rivers that were previously closed. King's Creek lesion prevalence in menhaden caught with cast nets in 1999 was 1.1% (n=1090). This declined dramatically from 1997 (22.0%, n= 4719) and 1998 (10.4%, n = 318). Cast netting was not done in the Pocomoke River in 1997, so data from all gears was combined for comparing the September through November time period. Pocomoke River menhaden lesion prevalence declined through the years from 77.2% in 1997 (n= 179), to 8.4% in 1998 (n= 321) and 0.0% in 1999 (n =63).

When comparing sites sampled at the same frequency in 1998 and 1999, total catch (seine and cast net) of menhaden increased more than 1300% in the Chicamacomico River and Kings Creek in 1999. At these sites on the Pocomoke River, in 1998 only 4 menhaden were captured compared to 23,006 in 1999. Menhaden catches increased by 1917% in Fishing Bay, almost 300% in the Nanticoke River, 70% in the Annemessex River and 49% in the Manokin River. The 1999 Wicomico River menhaden catch decreased by 353% because of a single unusually large seine haul in 1998 (27,770 menhaden).

In 1998 over 96% of the menhaden captured were at temperatures between 23.0-28.9C and 64% fell within the same range (23.0-28.9C) in 1999. D.O. between 8 and 9.9 mg/l accounted for over 93% of the menhaden captured in 1998 and 41% in 1999. In 1999, 77% of the menhaden were found between 6.0 and 9.99 mg/l. In 1998, over 85% of the menhaden captured were found in salinities of 6.0-7.9 parts per thousand (ppt). In 1999, four different salinity ranges each accounted for more than 10% of the total menhaden captured. Nearly 40% were collected in 12.0-13.9 ppt in 1999.

Temperatures of 27.0-28.9C accounted for 59% of menhaden with anomalies in 1998 and 39% in 1999. In 1998 nearly 92% of all menhaden collected with anomalies were found in a dissolved oxygen range of 6.0-9.99 mg/l. In 1999, 89% of menhaden with an anomaly were collected at 6.0-9.99 mg/l. During 1998 and 1999, 35% of menhaden with anomalies were captured at 10.0-11.9 ppt. In 1998 one sample produced over 27,000 Atlantic menhaden with 10 anomalies. This sample weighs heavily in the 1998 percentages.

The Fish Health Hotline received 202 calls in 1999. A large proportion (28%) of the calls concerned striped bass displaying shallow sores and reddening of the skin. These calls comprised 35% of 441 calls in 1998. These reports were clustered in areas of high angling activity and were made during the striped bass open season. Most of the remaining calls were from isolated incidents throughout the state involving 15 different species.

Fisheries Service and Department of Environment Fish Kill Unit biologists responded by phone or personal contact to 109 citizens that contacted the Hotline. In most cases, samples of fish were collected by responding biologists and diagnoses were attempted. Several calls reported menhaden with lesions and Rapid Response Teams were sent to collect menhaden and determine the proportion of lesioned fish. None of the sampling resulted in river closures.

On July 22, 1999 a response was made to a report of an Atlantic menhaden kill in Pocomoke Sound. Approximately 500,000 menhaden succumbed to low dissolved oxygen in Bullbegger Creek, a Virginia tributary to Pocomoke Sound. The fish appeared otherwise healthy and had been swept into the Sound by the tide. No living menhaden were captured during the initial investigation. However, 503 healthy menhaden were captured on July 30 and no subsequent problems occurred in this area.

The report of a kayaker becoming ill after exposure to the water of Back Creek, a tributary to the Manokin River, resulted in 9 days of fish sampling during August 11- September 14, 1999. Only 1.8% of 2178 menhaden captured had anomalies. Forty-two fish of other species were captured and all were healthy. No further human health problems occurred and sampling was discontinued.

A response to a call to the Fish Health Hotline involving menhaden with ulcers in Middle River was made on August 22, 1999. Sampling was conducted on 13 days during August 22 - September 22. We captured 1698 Atlantic menhaden and 63.2% (1073) had anomalies; 83% of anomalies were ulcers. Ten other species were captured (n=710), with an anomaly rate of 0.3% (2 fish). During the last 2 days of sampling only 54 menhaden were captured, with an anomaly rate of 20%. Four other upper Chesapeake Bay tributaries were sampled periodically from August 29 to September 30, 1999. The Patapsco and Back rivers produced small catches of menhaden with anomaly rates approximately 15%. The Gunpowder River sampling captured 56 menhaden with a 68% anomaly rate. Sampling conducted on the Magothy River produced 167 menhaden with only 1 nonulcertive lesion (0.6%) recorded.


References

Lukacovic, R., H. Rickabaugh Jr. and H. Speir. 1999. Comprehensive Fish Health (Lesion) Sampling - 1998. Maryland Department of Natural Resources Fisheries Technical Report Series no. 26. Annapolis, MD.

Noga, E.J., L. Khoo, J.B. Stevens, Z. Fan, and J.M. Burkholder. 1996. Novel Toxic Dinoflagellate Causes Epidemic Disease in Estuarine Fish. Marine Pollution Bulletin, Vol. 32, No. 2, pp. 219-224.

Speir, H. and B. Pyle. 1998. A Survey of Distribution and Prevalence of Fish Lesions in Chesapeake Bay, Maryland, 1997. Maryland Department of Natural Resources Fisheries Technical Memo no. 13. Annapolis, MD.






Summary: Pocomoke Watershed Pollution Assessment 1999



Prepared by:



Maryland Department of the Environment

Maryland Department of Agriculture

United States Geological Survey

Maryland Department of Natural Resources

Natural Resources Conservation Service

Wicomico Soil Conservation District

Worcester Soil Conservation District

Somerset Soil Conservation District



Introduction

The Pocomoke River watershed is a combined watershed that includes the drainages of the Pocomoke, Manokin and Big Annemessex Rivers. The Pocomoke River watershed drains approximately 433 sq. mi. of Worcester, Somerset, and Wicomico Counties in Maryland, 38 sq. mi. in Delaware, and 17 sq. mi. in Virginia. The primary tributaries of the Pocomoke River are Dividing (62 sq.mi.) and Nassawango(69 sq.mi.) Creeks. The Manokin and Big Annemessex Rivers drain 163 sq. mi. in Somerset and Worcester Counties, Maryland.

The land cover in the Upper Pocomoke River watershed and the Lower Pocomoke River watershed is forested and agricultural. Forest accounts for 53% of the land use in the Upper Pocomoke and 58% in the Lower Pocomoke. This is slightly less than the overall percentage of forest cover for Maryland's Lower Eastern Shore Tributary Strategy Basin, which is 61% forested. Agricultural land in the Upper Pocomoke accounts for 45% of the land use, while in the Lower Pocomoke it accounts for 36% of the land use. This is slightly higher than the overall percentage of agricultural land for Maryland's Tributary Strategy Lower Eastern Shore Basin, which is 32% agricultural. Urban land in these two subwatersheds accounts for 3% of the land use, while overall in the Lower Eastern Shore Basin, urban land accounts for 5% of the land.

The largest communities in the Pocomoke watershed are Pocomoke City, Princess Anne, Crisfield and Snow Hill.

Agriculture in the watershed is mixed cash grain, vegetable crops and livestock. Corn, soybeans, and small grains are the predominate crops. Poultry production is the main type of livestock agriculture. The annual poultry population is estimated at approximately 22,400,000. The manure produced by all of the livestock in the watershed is estimated to be in excess of 329 million pounds.

The modeled nutrient loads for these two basins are not proportional to the land use (1996 CBPO Phase IV Chesapeake Bay Watershed Model Scenario)( MD DNR ,1999). In the Upper Pocomoke basin, 90% of the total nitrogen load is from agricultural land, followed by forested land at 6%, urban land at 2% and point source loads at less than 1 percent. Sources of loads in the Lower Pocomoke are similar to the Upper Pocomoke with 82% of the total nitrogen load from agricultural land, 8% from forested land, 3% from urban land, and 3 % from point source loads. Loads from septic systems and atmospheric deposition to open water make up the difference. For total phosphorus loads, 80% of the load is from agriculture in the Lower Pocomoke, and 97% of the load is from agriculture in the Upper Pocomoke.

In an assessment of the 138 Maryland 8-digit watersheds (of which the Lower Pocomoke River and Upper Pocomoke River are two), a variety of indicators were examined in order to prioritize watersheds for either restoration, protection, or both (Maryland Clean Water Action Plan Technical Workgroup, 1998). These indicators covered water chemistry, aquatic living resources, and landscape characteristics. Both the Lower Pocomoke River and Upper Pocomoke River watersheds were determined to be in need of restoration. The Lower Pocomoke River watershed was also determined to be in need of protection.

In the Upper Pocomoke River watershed, 7 restoration indicators (of 11 pertaining to the watershed) were cause for concern. The most notable of these is the indicator entitled historic wetland loss. This watershed had the most acres of historic wetland loss than any other watershed in the state. 80,903 acres of historic wetlands have been lost. Historically, wetlands covered more than 85% of the watershed.

In the Lower Pocomoke River watershed, 8 restoration indicators (of 12 pertaining to the watershed) were cause for concern. The most notable include the indicators entitled SAV Abundance, SAV Habitat, and Historic Wetland Loss. This watershed received a score of "most degraded condition" for SAV Abundance. In fact, all of the MD 8-digit subwatersheds of the Lower Eastern Shore for which there was data, received this score. This watershed also received a very degraded score for SAV Habitat. The Lower Pocomoke River watershed received the second worst score for the state for the historic wetland loss indicator. 71,922 acres of historic wetlands have been lost. Historically, wetlands covered more than 70% of the watershed.

The Atlas of Indicators that has been compiled for the Lower Eastern Shore is presented as Attachment A. The Atlas is coupled with a description of the Lower Eastern Shore Conservation and Restoration Action Strategy Program. The Indicators have been used to target this restoration program in three small sub-watersheds of the Pocomoke Watershed. These three sub-watersheds will be the focus of further assessment and targeting and the focus of the programs restoration activities.


Paired Watershed Study in the Upper Pocomoke

In 1994 the Wicomico Soil Conservation District asked the Maryland Department of Natural Resources to join in a cooperative project designed to demonstrate the effect of nutrient management and poultry litter management on water quality. The project was originally designed to look at the effect on nutrient management and animal waste storage on water quality. In 1997 the outbreak of Pfiesteria in the lower Pocomoke River changed Maryland's approach to nutrient management and presented an opportunity for this project to evaluate a more aggressive approach to nutrient management.

The Water Quality Act of 1998, which was enacted as a result of the 1997 Pfiesteria outbreak, assumes that basing nutrient inputs on the phosphorus needs of a crop where soils test beyond optimum levels for phosphorus fertility, will improve water quality. To test this assumption the project has undertaken an intense implementation and monitoring effort designed to demonstrate the effect on water quality of eliminating P inputs and implementing cover crops on all available cropland.

The project is using a paired watershed study design comparing two watersheds with similar land use and farming practices. Two subwatersheds to Green Run, a tributary to the Upper Pocomoke in Wicomico County, were chosen as the study site. Grab and automated composite water quality samples from a control watershed, in which farming practices remain unaltered, and a treatment watershed in which BMPs will be implemented, will be compared to determine changes to in stream nutrient concentrations and loads. The calibration phase of the project has been completed with the collection of baseline nutrient concentration and load data from both watersheds prior to any BMP implementation. Elevated nutrient levels are evident in both watersheds. Complicating factors in the comparison include dry/no flow conditions and water column anoxia.

The analysis of the calibration period results indicate that significant water quality improvements can be achieved with a 9% change in the difference between mean total nitrogen concentrations at the outlet of the treatment and control watersheds during the treatment period. A 58% change in the difference between mean total phosphorus concentrations at the outlet of the treatment and control watersheds during the treatment period will be needed to improve water quality significantly. The Upper Pocomoke Calibration of the Agricultural BMP Evaluation 1994-1998 report is presented as Attachment B.


Ground Water Quality

The surficial aquifer on the Pocomoke watershed has a high silt and clay. The sediments are poorly drained because of poorly incised steams and flat water-table gradients. In much of this area, drainage ditches have been constructed, which will act to lower the water table. In the area closer to the Chesapeake Bay in the Pocomoke River Basin, the water table is generally shallow and comprised of poorly-drained, low-permeability sediment. The majority of ground-water in surficial aquifers discharges to adjacent streams and is termed "base flow". The percentage of total stream flow contributed by base flow ranges from about 42 to 74% (Bachman and others, 1998). These data are based on using hydrograph separation techniques at 20 sites with stream flow data.

Nitrate concentrations in the surficial aquifers underlying the Pocomoke River basin generally range from <0.1 to 35 milligrams per liter, with about 10 percent of the samples above the maximum contaminant level of 10 mg/L N as NO3 set by U.S. Environmental Protection agency. The median concentration was 1.1 milligram per liter. These values are based on data collected from 1976 to 1990 (Hamilton and others, 1993).

The nitrate concentrations appear to be related to land use and the type of sediments in the aquifer and soils. The nitrate concentrations were elevated in agricultural and residential areas in zones where the aquifer consists of sandy deposits (Hamilton and others, 1993). The sandy composition of the aquifer promotes aerobic conditions under which nitrate is stable. In agricultural and residential areas underlain by clay and silt deposits and anaerobic water, effects of human activities are less evident. The silt and clay deposits can cause lower nitrate concentrations for several reasons (Hamilton and others, 1993). The anaerobic conditions promote denitrification. The high amounts of clay and silt can inhibit downward movement of fertilizers from the land surface into the water table and have abundant exchange sites for ammonia, which reduces the amount of nitrogen available to reach the water table.

The age of the ground-water can also affect the nitrate concentrations. Knowing the apparent age of ground water can help explain nitrogen concentrations and provide an estimate of how long a management practice may take to be effective. Based on existing data the apparent ground-water ages in the surficial Coastal Plain aquifers range from about 5 to 20 years (Focazio and others, 1998).

1999 results from the ground water investigation in the Paired Watersheds support these findings. Nitrate levels vary from spring to fall and are higher along shorter flow paths. At the three sites samples nitrate concentrations ranges from 68 mg/l to <0.05 mg/l. The higher levels always occurring in wells adjacent to ditches representing short lateral flow paths. The lower levels always occurred in the deeper wells in the ditches representing longer flow paths up welling under the stream. Age dating of the water indicates that the apparent age of the water being sampled ranges from 5 to 20 years old. The younger water being collected in the wells adjacent to the streams and the older water being collected in the deeper wells in the stream beds.

Phosphorus concentrations did not follow the same pattern. Concentrations were either the same in the older water as the younger water being sampled adjacent to the streams or the concentrations were higher in the older water. Concentrations as high as 1.13 mg/l were measured in the water under the North Fork of Green Run in the spring of 1999. (Attachment C.)


Nutrient Loads

Nutrient loads and yields have been calculated for four sub-watersheds in the Pocomoke River watershed. The nutrient loads are flow driven. Loads increase with increasing flow for all constituents.

Nutrient yields are the annual loads discharged from a watershed divided by the acreage of that watershed. The yields allow comparisons to be made from one watershed to the next.

Annual Average TN and TP Yields (lbs/acre/year) in the Pocomoke Watershed

Year

North Fork of Green Run

South Fork of Green Run

Nassawango

Willards

TN Yield TP Yield TN Yield TP Yield TN Yield TP Yield TN Yield TP Yield
1994 6.90 0.80 8.80 1.03

1995

5.55

0.27

10.08

0.62

1996 21.93 1.48 42.81 8.52
1997 11.98 0.65 18.89 2.50 2.34 0.717
1998 20.47 1.04 26.23 4.21 2.76 1.18
1999 10.83 0.42 10.38 4.30 2.10 0.78

8.28

0.62

References

Bachman, L.J., 1984a, The Columbia aquifer of the eastern Shore of Maryland, part 1--hydrogeology: Maryland Geological Survey of Investigations Report 40.

Bachman L. J., B. Lindsey, J. Brakebill, and D. S. Powars. 1998. Ground-Water Discharge and Base-Flow Nitrate Loads of Nontidal Streams, and Their Relation to a Hydrogeomorphic Classification of the Chesapeake Bay Watershed, Middle Atlantic Coast USGS Water-Resources Investigations Report (WRIR) 98-4059, 71 p.

Denver, J.M., W.J.Gangloff, and P.A. Vadas. 1998. Surface Water Quality and Monitoring in Two Farms in the Inland Bays Watershed:1995-1997. In Assessing the Impact of Agricultural Drainage on Ground and Surface Water Quality in Delaware: Development of Best Management Practices for Water Quality Protection. Final Project Report. University of Delaware, Delaware Geological Survey and United States Geological Survey.

Focazio M.J., L. N. Plummer, J.K. Bohlke, E. Busenberg, L. J. Bachman, and D.S. Powars. 1998. Preliminary Estimates of Residence Time and Apparent Ages of Ground Water in the Chesapeake Bay Watershed, and Water-Quality Data From a Survey of Springs. USGS Water-Resources Investigations Report (WRIR) 97-4225,75 p.

Frink, C.R., 1991. Estimating Nutrient Exports to Estuaries. J. Environ. Qual. 20:717-724.

Hamilton, P.A. and others, 1993, Water-Quality Assessment of the Delmarva Peninsula--Effects of Agricultural Activities on, Distribution of, Nitrate and Other Inorganic Constituents in the Surficial Aquifer, USGS Open File Report 93-40

Maryland Clean Water Action Plan Technical Workgroup. 1998. Maryland Clean Water Action Plan. MD Department of Natural Resources, Annapolis MD.

Maryland Department of Natural Resources. 1999. Analyzing and Tracking Nonpoint Source Pollution in Maryland. Final Report, FFY97 Section319h CWA. MD Depart. Of Natural Resources, Annapolis MD.

 Pfiesteria Links:  FAQ | Monitoring | Publications | Contact Us
Pfiesteria piscicida | Pfiesteria shumwayae

Search Maryland DNR

Search www.dnr.state.md.us


Restoration and Protection | Bay Grasses | Harmful Algae | Bay Monitoring
Bay Life Guide | Bay Education

Return to the Maryland DNR Home Page.
Your opinion counts! Take a
survey!