Summary of Maryland’s Pfiesteria-related 2001 Work Plans

Prepared by Maryland’s Pfiesteria Study Team
DRAFT- March 15, 2001

INTRODUCTION
The study plan developed by Maryland’s Pfiesteria Study Team for 1998, 1999 and 2000 took a comprehensive approach toward understanding the environmental impacts and causes related to the outbreaks of Pfiesteria, related organisms and fish health problems in Maryland’s waters. The work plans for 2001 continue this comprehensive approach with scales of work intensity again prioritized according to a four-tiered approach: Level 1 - Rapid Response, Level II - Assessments of affected waterways, Level III - Assessments of similar waterways to those systems previously affected, and Level IV - Integration with existing monitoring programs of Baywide characterization. Level I objectives focus on the rapid response to assess suspected outbreaks of toxic Pfiesteria-like organisms and fulfill the State’s obligation for data needed in the protocol for river closures and reopenings. Comprehensive assessment objectives focus on increasing our understanding of the habitat links with toxic outbreaks and the vulnerability of areas to outbreaks, identifying HAB species and their distribution in Maryland waters, tracking improvements in water quality of impacted systems under implemented management plans, examining alternative hypotheses for observed fish health problems, and providing rigorous exposure data to epidemiologists studying the human health impacts.

OVERVIEW
Level I assessments will continue to support rapid response to fish health events as warranted. The Fish Health Hotline will again be in operation and incoming calls will be evaluated for any need of a rapid response to a described event and location. With the advances in applications of new molecular probes developed over the past year, the presence of Pfiesteria piscicida and several related species have been available within 24-48 hours of a sample collection. Samples of fish will be provided as warranted to the Sarbanes Oxford Laboratory and associate agencies for histopathological and microbiological analyses. Water samples will continue to be collected for harmful algal bloom assessments related to an event and comparisons with non event periods and locations. Samples will also be collected and testing conducted to define nutrient and other habitat conditions at an event to examine all potential factors affecting fish health.

Level II and III waterways will continue to serve as comprehensive assessments of rivers affected in 1997, 1998, 1999, and 2000, along with river systems with similar water quality and habitat conditions. Water and habitat quality sampling intensity will be biweekly sampling, April through October, on the Chicamacomico River and Pocomoke River in order to capture the temporal variability in key parameters such as nutrients, phytoplankton and dissolved oxygen. Sampling will be monthly on the Middle River, Manokin River, Nanticoke River, Wicomico River, Big Annemessex River, Trappe Creek and St. Martin River. Three water quality and fish sampling sites have been added to Ayer Creek, a tributary of Trappe Creek, and one water quality and fish sampling site has been added to Marshall Creek in response to lesion outbreaks in 2000. These new sites will be sampled twice a month. Longitudinal profiling for in vivo fluorescence will continue in 2001 to document the spatial and temporal positioning of chlorophyll maxima in each of the nine monitored systems, following the same frequency for each system as described above for water and habitat quality sampling. Phytoplankton species composition data will be collected at selected locations along with selected additional chemical analyses to discriminate dissolved organic fractions (e.g. urea). Baseline sediment surveys to map the distribution of Pfiesteria will continue, along with associated chemical/physical characteristics, but the analysis of Pfiesteria will now use PCR techniques which were initiated in the fall of 2000.

Fisheries is proposing to discontinue biweekly sampling on the approximately 11 systems surveyed May-October, (Levels II and III). Sampling is planned to continue with surface trawls, cast netting and seining for menhaden on the Pocomoke River in 2001. A subset of the fish health surveillance stations will also be used as simultaneous water quality and Pfiesteria survey sites to better understand the associations between fish health, Pfiesteria and water quality.

Experimental work on ulcer progression of myxosporidian infections and their possible role in chronic ulcerative lesions in menhaden will be examined. Cage studies at sites of potentially toxic Pfiesteria may also be employed during this study year.

Level IV Baywide characterizations (enhancements and utilization of existing programs) will continue to provide essential data for comparisons and links to the intensified data collections that are occurring on the Lower Eastern Shore region and Middle River. Details of the program work plans are summarized in the following documents.

Analysis of data collected since 1997 will move toward integration of water quality, habitat Pfiesteria, the algal community and fish. Hypothesis testing will follow the objectives established at the outset of the program including association between Pfiesteria and environmental factors, and Pfiesteria and fish health.

The year 2001 will most likely represent the last year during which data will be collected with the expressed purpose of evaluating the relationships between Pfiesteria and fish health. In 2002 and beyond the program will continue to satisfy rapid response objectives, evaluate relationships between Pfiesteria and other HABs and environmental conditions, track the effectiveness of management actions aimed at lowering nutrient impacts, provide data to human health researchers, and analyze the cumulative data sets to satisfy stated objectives.

Table of Contents for Maryland’s Pfiesteria-related 2001 Work Plans

MARYLAND’S PFIESTERIA-RELATED 2001 WORK PLAN:
HABITAT QUALITY AND PFIESTERIA
DRAFT- 3/15/01

Tidewater Ecosystem Assessment
Maryland Department of Natural Resources
580 Taylor Avenue
Annapolis, MD 21401

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 . 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. Sampling in 1998 and 1999 has shown that Pfiesteria continues to be found active in the water column at the sites of major menhaden lesion outbreaks and in the sediments of a number of other tributaries. 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 2001 sampling of habitat quality and Pfiesteria surveillance are the following:

A. Rapid Response

1. Provide for the rapid response testing of, and reporting on, habitat quality and Pfiesteria-like organisms in cases of suspected toxic outbreaks.
2. Provide fish and water quality data needed to fulfill DNR’s obligations under Maryland’s
“Protocol for Closing and Reopening Rivers Affected by Pfiesteria or Pfiesteria-like Organisms.”

B. 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.

STUDY DESIGN AND METHODOLOGY

A. Habitat Quality Monitoring

Rapid response (Level I)
When a rapid response team is unable to identify an apparent cause of the fish health problem (such as hypoxia, chemical spill, etc.), or when instances of human health problems meeting the Centers for Disease Control criteria are reported, three water quality samples will be collected as soon as possible; one at the center of the event site, and one each upstream and downstream of the center of the event within the event site.

Longitudinal sampling (Level II and III)
Continuation of longitudinal sampling initiated in 1998, 1999, and 2000 at 8-17 stations April through October on the following rivers:
Level II: Pocomoke (twice a month), Chicamacomico (twice a month), Manokin (once a month).
Level III: Middle, Nanticoke, Wicomico, Big Annemessex, St. Martin, Trappe Creek/Newport Bay (all once a month).

Long-term water quality monitoring (Level IV)
As part of the Chesapeake Bay Water Quality Monitoring Program, DNR will continue to sample water quality at 55 stations in the Maryland tributaries and 22 stations in the Maryland mainstem once or twice a month (depending on season).

Measured variables
Sampling under all levels (Level I-Level IV) includes a broad suite of physical and chemical variables. Several variables (specific conductance, temperature, dissolved oxygen, pH, and secchi depth) are measured in situ; discrete water samples will be collected and returned to the laboratory for analysis of additional variables, including nitrogen, phosphorus, carbon and silicon species, total suspended solids and chlorophyll a. Due to little, if any, consistent differences with depth in areas of low vertical density gradients, generally only a surface sample will be taken for nutrients for Level I, Level II or Level III systems. In Level IV systems, samples will be taken according to the long-term monitoring program protocols, which include samples taken at the surface and bottom, and at the deeper stations where stratification is an issue, above and below the pycnocline.

Data management and analysis
Data will be managed using the same procedures in place for the Chesapeake Bay Water Quality Monitoring Program. Data needed for rapid response investigations and to determine closing and openings of rivers will be prepared in simple tabular formats within hours of receipt from the field and laboratories.

For comprehensive assessments in Levels II and III systems, various exploratory analyses will be used to help identify spatial and temporal patterns in prior years. Historic data will be used when available to draw comparisons between current conditions and those in the past. The analysis of various parameters will be conducted with the goal of satisfying study objectives. Analyses will include:

1. Regional water quality patterns for Level II and III tributaries of the Chesapeake and Coastal Bays
2. Current status and long-term trends for Level II, III and IV systems
3. Comparison of nutrient and habitat quality conditions among tributaries within the appropriate salinity regime
4. Space-time distribution of habitat measures
5. Precipitation and river flow
6. Salinity dilution plots
7. Analyses of nutrient pools to understand patterns and transformations between different nutrient fractions

In addition to the exploratory analyses, univariate and multivariate methods will also be used in comparisons of affected vs. unaffected regions to objectively identify water quality and other variables that may be critical in identifying vulnerable water bodies and also suggest potential remedial measures.

B. Pfiesteria piscicida, related dinoflagellate and associated algal community monitoring

Rapid Response (Level I)
When no other cause for the observed fish health problem is apparent, or human cases of illness meeting the CDC criteria are reported, Rapid Response Teams will collect three water column samples; one at the site of the event, and one each upstream and downstream of the epicenter, but still within the extent of the event. If rapid response evaluations result in the closure of a river, water samples for Pfiesteria and associated phytoplankton will continue to be collected daily for the first three days of the event, and then every third day thereafter until the river is re-opened. Sampling site locations may change and expand as the epicenter of the event moves.

Collected water samples from rapid response events will be split among three primary laboratories. Preserved water column samples will be scanned under light microscopy by Dr. Burkholder’s laboratory (NCSU) for Pfiesteria piscicida-like cells; if presumptive P. piscicida cells are found, fish bioassays will be conducted with unpreserved samples to determine toxicity; if the bioassays are positive, scanning electron microscopy will be conducted to confirm species identification. Unfixed samples will also be sent to Dr. Oldach’s laboratory (UMD) for detection of Pfiesteria’s presence through the use of molecular probes. Finally, fixed water column samples will be analyzed by the Academy of Natural Sciences for composition of associated algal community to the lowest practical taxa. Selected samples will also be sent to Dr. Steidinger’s laboratory (FL DEP) for SEM, Dr. Rublee (UNC Greensboro) and Dr. Vasta (UMD COMB) for PCR analysis, and other researchers as appropriate.

Comprehensive assessment (Level II and Level III)
Water column samples for dinoflagellate and phytoplankton species composition will be collected twice a month at three stations in the Pocomoke River and Chicamacomico River in areas of outbreaks of Pfiesteria activity in 1997 and/or areas of high chlorophyll densities. The Middle River, Manokin River and St. Martin River also will be sampled once a month at three stations. Once a month sampling at three stations in Ayers Creek and one in Marshall Creek have been added in response to lesion fish outbreaks in 2000. Microzooplankton samples will be collected at one station in each of the Pocomoke, Transquaking, and St. Martin River and at the station in Marshall Creek.

After settling, preserved samples are initially enumerated at 500X using a random fields technique; a minimum of twenty random fields and 200 individual cells are enumerated and additional fields are counted until the minimum count is attained. A low magnification scan (312.5X) is then done of 20 random fields to estimate the rarer, larger forms within the sample.

Individual taxon cell volumes are determined by calculating the dimensions of the cell, determining the appropriate shape of the cell and applying the dimensions to the volumetric equation (or combination thereof) for that particular shape. The cell dimensions are calculated by two methods - direct microscopic measurement and assigning a mean value from the dimensions cited in the literature. Cell carbon values are generated from literature values and estimates based on cell total area and volume.

Data management and analysis
Rapid response sample presumptive counts results will be communicated as soon as possible via telephone, fax, or e-mail to DNR staff and entered into a spreadsheet table for tracking during extended investigations. When bioassays are necessary, results will be submitted as summary reports to DNR staff, who will keep a catalog of all results, both toxic and non-toxic. As needed, interpretation of results will be based on consultations with the Technical Advisory Committee.

Species composition data will be provided to DNR in Quattro Pro spreadsheet files which will be converted into SAS files to become part of the comprehensive database of SAS files constructed for coordination of deliverables with the Chesapeake Bay Program. Level II and III system species composition data will be interpreted regarding changes with season in dominant species (both numeric and biomass dominants), the presence and abundance of dinoflagellates and other key species (including Cryptomonas sp. thought to be preferred food for Pfiesteria and other species that may be important food for menhaden), linkages with nutrients and comparisons to 1998, 1999, 2000, and 2001 data and to data from the Chesapeake Bay Long-term Phytoplankton Monitoring Program (which includes data back to 1985 for other areas around the Bay and its tributaries).

C. Molecular Probes

During rapid response events, DNR will collect water samples from affected systems for testing of new molecular technologies (e.g. toxin and molecular probes) under development by Dr. Rublee, Dr. Oldach and others. Samples will be processed according to each laboratories protocols and requirements.

DNR will also be collecting monthly water samples from Level II, Level III and Level IV systems to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae by Dr. David Oldach’s laboratory at University of Maryland. This work will identify presence/absence of Pfiesteria and related cells using DNA probes developed by Dr. Oldach and will be used to determine areas that have Pfiesteria populations (note: alone, this work will only tell presence/absence, not whether the cells are or could become toxic). A subset of the fish health surveillance stations will also be used as simultaneous water quality and Pfiesteria survey sites for improved linkage of analyses between the three component programs.

D. Supplemental sampling (all supplemental sampling is contingent on the availability of funding. Funding levels for 2001 have been significantly reduced. It is therefore critical to evaluate the importance of each supplemental sampling component)

1. In vivo Fluorescence
Horizontal distributions of chlorophyll a will be determined along the longitudinal gradient of all Level II and III systems up to the tidal fresh water segment. These measurements will allow better determination of location and extent of peak chlorophyll areas linked to phytoplankton blooms and allow for tracking of seasonal changes in the location, extent and dominant species in these blooms. Continuous measurements will also identify bloom locations that do not coincide with fixed sampling locations. Profiles will be done once a month (twice a month in the Pocomoke and Chicamacomico Rivers) from May through September, using in vivo fluorescence. Samples for phytoplankton species composition (see above) will also be collected from within the peak bloom areas. Sampling and analysis will be done by the Academy of Natural Sciences.

2. Urea
Elevated concentrations of urea have been associated with high concentrations of Pfiesteria (Lewitus et al. 1999). Samples will be collected from 6 sites each from the Pocomoke (twice a month), Chicamacomico (twice a month), Manokin (once a month) , St. Martin (once a month) and Middle River (once a month). Samples will be analyzed and reported on by Horn Point Laboratory, Cambridge, MD. Urea will be analyzed using the urease method (Parsons et al. 1984).

Graphical analysis will be used for exploratory analysis. The urea data will also be incorporated into the broader data analysis effort to relate nutrient conditions to Pfiesteria and phytoplankton species composition.

3. Sediment and Water survey
Sediment sampling will be conducted to test sites for the presence of Pfiesteria and Pfiesteria-like organisms and to characterize the sediment-nutrient environment. Sediment samples will be sent to both Dr. Oldach’s and Dr. Rhublee’s laboratories for processing using molecular probes.
Samples sites will be determined based upon analysis results from 1998-2000 work, and will be randomly selected from all areas of these systems that meet appropriate salinity and depth criteria.

Maryland Geological Survey will collect surficial sediment samples (top four centimeters); sediment will be subsampled and analyzed for water content, porosity, bulk density, and grain size according to MGS standardized techniques. The dried sample from the water content analyses will then be sent to the Chesapeake Biological Laboratory for analysis of particulate nitrogen, particulate phosphorous and particulate carbon

Approximately 100 unfixed water samples will also be collected from approximately 70 Level II, III, and IV sites per month and sent to Dr. Oldach’s laboratory for detection of Pfiesteria’s presence by molecular probe. Samples will be collected at established sites and will be coordinated with corresponding water quality and algal community whenever possible. The objective of this sampling is to explore the spatial and temporal distribution of Pfiesteria in Maryland waters and the associated habitat conditions.

4. Continuous monitoring
A continuous monitoring project will be continued in 2001. The primary objective of the continuous monitoring work is to provide time-relevant data near the site of 1997's Pfiesteria outbreaks on the Pocomoke and Chicamacomico Rivers and another site on the Transquaking River to classify conditions and time scales over which physical, chemical and biological processes occur in this estuary. A second objective is to evaluate summer hypoxia (low dissolved oxygen) which develops in the middle portions of the Pocomoke River. In 2001, monitoring stations will be divided between 3 sites in the Pocomoke River with 2 additional sites in the Chicamacomico/Transquaking drainage.

High-frequency (every 15-min) observations of water temperature, salinity, turbidity, pH, fluorescence and dissolved oxygen will be collected using a YSI6600 moored in place from May through October. Weekly water samples will be also collected to measure nutrients, chlorophyll a and water column respiration rates.

The high frequency observations on dissolved oxygen and physical conditions will be combined with water column respiration rates to study the principal causes of summer hypoxia (work is in progress).

References

Lewitus et al. 1999.

Parson, T., Y. Maita and C.M. Lalli. 1984. A manual of chemical and biological methods for seawater. Pergamon Press, Oxford.


MARYLAND’S PFIESTERIA-RELATED 2001 WORK PLAN:
FISH POPULATION, FISH HEALTH AND LESION SAMPLING
DRAFT 3/15/2001

Maryland Department of Natural Resources
Fisheries Service: Biological Monitoring and Analysis Program and Sarbanes Cooperative Oxford Laboratory
Resource Assessment Service: Fish Index of Biotic Integrity Project

University of Maryland Center for Environmental Science
Horn Point Environmental Laboratory

In Cooperation with
Maryland Department of the Environment: Fish Kill Investigation Unit

INTRODUCTION
Maryland has adopted criteria to close bodies of water which may be affected by harmful algal blooms (HAB’s). Closure criteria depend on the determination of a toxic Pfiesteria outbreak based upon fish health and mortality, presence of Pfiesteria in sufficient densities, the absence of alternative causes for fish mortality events and appropriate habitat conditions. The Department of Natural Resources’ (DNR) Fisheries Service initiated a program in 1997 to monitor closed tributaries for the presence of ulcerated fish. These directed programs were expanded in 1998, 1999 and 2000 to 1) sample Chesapeake Bay tributaries considered at risk because of similar environmental characteristics, and 2) conduct experimental studies directed at determining the etiology of the ulcerative lesions found in Atlantic menhaden (Brevoortia tyrannus). Also starting in 1998, all fish collected in other estuarine fish monitoring projects were examined for the presence of exterior lesions. In Maryland tidewater the prevalence of anomalies among all species was 0.54% (n=372,675) in 1998, 0.46% (n=347,081) in 1999 and 0.97% (n=344460) in 2000.
Observations of fish collected during the morbidity events in 2000 reaffirmed the role of fungal infections in lesion development, but also indicated that myxosporidians (protistan pathogens) could initiate chronic ulcerative lesions in juvenile menhaden.
Several field experiments with caged fish were completed by Horn Point Environmental Laboratory (HPL) during fish morbidity events in 1999. The success of these experiments has provided a basis for more focused studies in 2000.

OBJECTIVES

1. Provide information on the incidence and distribution of external anomalies on fish in Maryland’s portion of Chesapeake Bay and oceanside tributaries.

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

3.Through coordinated experimental studies, field observations and focused pathological investigations, pursue a goal of determining the principal factor or factors involved in initiating ulcerative disease in Atlantic menhaden.

4. Perform an experimental study to document progression of myxosporidian infections and their possible involvement with the chronic ulcerative lesions in menhaden.

5. Respond to citizen calls to the DNR Fish Health Hotline.

6. Provide researchers with healthy and diseased fish for histopathological and microbiological analysis.

7. Work cooperatively with Maryland Department of the Environment (MDE) to investigate fish morbidity and mortality events.

STUDY DESIGN AND METHODOLOGIES

A. Fish Studies

1. Field Monitoring

Rapid Response

i. STUDY DESIGN:
A 24-hour fish health hotline will be maintained by the Natural Resources Police and based in the Tawes Building in Annapolis. Biologists in the Biological Monitoring and Analysis Program (BMAP), Sarbanes Cooperative Oxford Laboratory (SCOL) and Resource Assessment Service (RAS) will cooperate with Maryland’s Department of the Environment’s (MDE) Fish Kill Project to constitute the Rapid Response Team. The team will respond to potential problem areas identified through the Hotline. A cooperative working agreement between MDE’s Fish Kill Project, SCOL, RAS and BMAP specifies responsibilities and duties of the biologists responding to potential or active fish kill reports or lesion events.
An after-hours and weekend response capability will be activated beginning May 1 and continue through October. Personnel from MDE, BMAP, Fish Health Project and RAS will be on standby to respond to Hotline calls of fish kills or lesion events.

ii. SAMPLE ANALYSIS
Dead or dying fish at the site will be documented, identified by species and enumerated using established fish kill counting guidelines. Fish will be sampled at the site of the reported incident using gear most appropriate to the physical characteristics of the water body and the situation. Fish samples will be retained and processed for histology, microbiology or toxicology if they are suitable for diagnostic purposes. Water samples to detect HAB’s, habitat quality and pollutants will be taken along with temperature, salinity, pH, dissolved oxygen and secchi depth when dead, dying or lesioned fish are present or closure criteria are met.


iii. DATA MANAGEMENT AND ANALYSIS
Project biologists are trained in boat handling and sampling protocols. Sampling techniques and data recording practices will follow those outlined in the Field Sampling Manual for Collection of Fish Data in Support of the Pfiesteria Monitoring Project and the MDE Fish Kill Investigation Manual. Gear will be deployed in a consistent manner to assure comparable catch per unit of effort (CPUE) from river to river and year to year. Personnel are trained in fish species identification, lesion classification, necropsy techniques and water quality sampling. Standardized field sheets are used to facilitate data entry. Coordinates of area sampled will be recorded using the NAD 27 coordinate system. Trained personnel will enter fish data into an Access data base in an establish, consistent format. Edit checks will put limits on parameters to eliminate errors. The data format is easily imported into statistical packages such as d-Base, SAS, SPSS and GIS systems. Printouts will be printed, verified and checked for outliers. A copy of all data collected will also be imported into the Fish and Wildlife Health Program database at SCOL. Data will be examined for time series trends and correlations with other environmental parameters.


Baywide Characterization
Within DNR, six Fisheries BMAP projects and one RAS project will monitor specific populations of fish and crabs in Chesapeake and oceanside rivers and bays. (Some of these water bodies were previously listed under Levels II or III. Sampling methods for Level IV are different than Levels II and III and target different fish species and habitats.)

For baywide characterization sampling, all Fisheries Service and Resource Assessment Service projects will note the frequency and type of anomalies on fish collected in various fishery-dependent and fishery-independent samples taken in Maryland tidewater. Fishery-dependent samples from commercial and recreational fisheries will include pound nets, fyke nets, gills nets, eel pots and hook and line. Fishery-independent methods include otter trawls, beach seines and cast nets. Projects providing data include an angler survey on head boats out of Crisfield, the Striped Bass Juvenile Survey, Striped Bass Tagging Program, Blue Crab Trawl Survey, Bay Pound Net Survey, Coastal Bays Trawl and Seine Project, Baseline Fish Health Survey, Biological Indicators Trawl Survey and Fish Index of Biotic Integrity Project. An additional multispecies investigation trawl survey in cooperation with the Univ. of Md. Will be started this summer. Sampling location and frequency have not been worked out but the survey will provide additional coverage to these listed rivers as well as expanded coverage into other rivers.


2. Research
i. LESION PROGRESSION
In 1999 and 2000, post-metamorphosis menhaden were captured in the Pocomoke River with external lesions ranging from small areas of reddening to penetrating dermal ulcers. In both years, lesions first appeared in May-June and were absent by early July. In Pocomoke River, as well as other tributaries, lesions characterized as “ulcerative mycosis” have been observed from July into September. Spores of the myxosporean Kudoa clupeidae have been found in most menhaden microscopically examined throughout the May-September sampling season. All May-June lesions have been associated with an unidentified invasive plasmodium. It is currently unknown if these “early lesions” are associated with ulcerative mycosis. In 2000, an observational study conducted at the Horn Point Laboratory of fish with these “early lesions” resulted in the complete disappearance of the lesions within one month and subsequent low mortality. This observation suggests the fish were able to heal although minimal histological evidence of healing was observed. Some of the deceased fish had significant lesions associated with the invasive plasmodium in their visceral organs. However, no evidence of the invasive plasmodium was apparent in any fish after healing. The unexpected speed with which the lesions healed precluded an opportunity to observe the progression of disease and gain an understanding of the mechanism by which menhaden healed. In 2001, seine sampling will be conducted by HPL to follow the progression of disease if and when early lesions are detected by DNR sampling crews. Samples of healthy and clinically infected juveniles will be transported to HPL and held in separate flow through tanks grouped by lesion severity as follows:

Control: No grossly observable abnormalities
Mild: Minimal diffuse hemorrhage to moderate focal hemorrhage in the epithelium
Severe: Raised focal or multi-focal protrusion of the epithelium

Each categorical group will be sub-sampled weekly or bi-weekly depending on the actual size of the overall sample. Sub-samples will be processed for routine histology and histopathological observations noted . Through this effort, we hope to further our understanding of the relationship of Kudoa clupeidae and the unidentified plasmodium with ulcerative conditions in menhaden.


ii. KUDOA sp. PCR PROBE
Surface trawls will be used from April through the end of May in the Pocomoke River to capture post metamorphis menhaden. Surface trawls are 8 ft x 8 ft nets of 3/8 in stretch mesh lined in the codend with 500F plankton netting. They will be towed for six minutes with the tide. Seine hauls will be made from the beginning of May through the end of July on the Pocomoke River. Seine hauls will be made using a 100 ft x 4 ft net of ½ in stretch mesh. Collections will be made at historic sampling locations.
Tributaries will be sampled at specific access points using cast nets from May through October. Twenty casts will be made with a 6 ft net (1 in stretch mesh) at each designated site. Cast net sites will be accessible by land. All sampling on the Pocomoke river will be done on a biweekly basis.
Catch will be counted by species. Any abnormalities such as lesions, mechanical damage or parasites will be noted. Lesions will be described as new, old or recovering. All individuals with anomalies will be measured. Collections of lesioned juvenile menhaden for Kudoa sp. PCR analysis will be collected. Two sets of 20 fish with mild lesions and 20 fish with severe lesions will be collected. One set will be frozen on dry ice, and the second set of fish will be fixed in paraformaldehyde. Ten additional fish will be prepared for electron microscopy using gluteraldehyde. Sub-samples of additional menhaden (5-10 depending on size) will be fixed whole, or necropsied as appropriate, and fixed in 10% neutral buffered formalin for histological examination. These collections may be made each day a particular tributary is sampled, however, collections will be prioritized according to the needs of cooperating pathologists and the specific questions to be addressed. Multiple fish without external lesions can be fixed in one container, however fish with lesions will be fixed in individual containers. These containers will be labeled with the date, river, site, and corresponding fish number from the data sheet. Samples for microbiology will be taken at the request of fish health specialists.

iii. CAGE STUDIES
From 1997-2000 HPL and University of Maryland Eastern Shore U.S. Fish and Wildlife Service Cooperative Unit have been developing techniques for caging white perch (Morone americana) and juvenile menhaden. The principle of these studies was to place known healthy fish in an environment where fish kills or lesions were found and follow the progression of ulcer formation. We have been successful caging both species with minimal stress, but no ulcer formation has been noted in two years of study. It is apparent from 1999 caging efforts on the Middle River that using this assay after lesions are detected is not a worthwhile endeavor. Ulcerated fish were captured from the wild at caging sites, while those in the cages remained unaffected. We have demonstrated that it is possible to transport juvenile menhaden across the state and cage them for over two weeks with minimal impact. Therefore, cages will be used in 2001 only under the following criteria:

Pfiesteria-like organisms (PLOs) are detected at potentially toxic levels by means of presumptive counts, or an acute fish morbidity or mortality event is observed. Acute events are defined as either a fish kill in progress or large numbers of fish displaying erratic behavior.

MARYLAND'S PFIESTERIA-RELATED 2001 WORK PLAN: WATERSHED POLLUTION SOURCE ASSESSMENT
DRAFT- 3/15/2001

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 watershed pollution source assessment involves a multi-faceted study plan designed to provide the information and assessment tools required to estimate the contribution of all major pollution sources in the Pfiesteria affected watersheds and to evaluate the effectiveness and time-frame of expected water quality response of ongoing pollution control efforts. The study involves four major components:

(1) characterization of pollutant sources through inspection and monitoring,
(2) estimation of pollutant loads through continuous monitoring and modeling,
(3) evaluation of management practices through paired watershed monitoring,
(4) identification of pollutant transport pathways and the timing of pollutant delivery to surface waters.

OBJECTIVES

1. Characterize the relative magnitude of pollutant sources overall and for different portions of the watershed.
2. Estimate pollutant loading from sources and areas identified above.
3. Estimate the effectiveness of management practices in reducing pollutant loading to the affected areas.
4. Determine transport pathways and estimate "lag time" of pollutant delivery.
5. Provide watershed characteristics to user groups such as the Lower Eastern Shore Tributary Team to help direct implementation efforts.

STUDY DESIGN AND METHODOLOGIES

A. General Overview.


During 1997 and 1998 point and nonpoint source water quality monitoring was initially conducted in the watersheds that have been previously closed due to Pfiesteria outbreaks (Pocomoke, Kings Creek and Chicamacomico). Starting in 1998 monitoring activities have been enhanced in the Pocomoke watershed and expanded to include other similar watersheds, moving from south to north on Maryland's Eastern Shore, including the Coastal Bays area.

Discharge and water quality data will continue to be collected in 2001 at all regulated point sources and at representative stream/river sites in the study watersheds. A network of monitoring sites on all major tributaries in the study watersheds will continue to be sampled periodically during both high and low flow periods to obtain a broad characterization of water quality conditions. Two new sites were established in 1998, bringing to a total five sites to provide both continuous flow monitoring and water quality monitoring in the Pocomoke watershed. These sites will continue to operate during 2001. The broad array of grab sampling sites in combination with the continuously monitored sites will continue to provide the data needed to document both the spatial and temporal variation of pollution sources in the watershed. Water quality monitoring primarily focuses on nutrient, sediment and bacteria levels. Monitoring for pesticides and other potentially toxic contaminants will continue to be added periodically to screen for possible contamination and/or if specific contaminants of concern are identified during inspections of permitted facilities or agricultural operations.

In conjunction with the water samples, data on watershed characteristics (land use, soils, slopes, etc.) and land management practices (crop types, fertilizer, manure, sludge application, and other data) will continue to be compiled in GIS format in order to support the development of computer models of the watershed, river and estuary. The models will be calibrated to the available water quality monitoring data. The models will be used to estimate the relative pollutant loading contributions from different portions of the watershed and from different source areas (cropland, urban/residential, forest, wetlands, etc.) and sources (point sources, onsite sewage systems, manure, fertilizer, sludge, etc.). This information is needed to guide pollution control efforts into the areas and to the sources that are the most significant contributors of the pollutant(s) of concern.

Paired watershed sites are being used to monitor the effectiveness of selected management practices appropriate for the Lower Eastern Shore. The project is a nested paired watershed study looking at the effect of an aggressive conservation program on nutrient concentrations and loads being discharged from the Green Branch sub- watershed into the Pocomoke River. The paired watersheds are nested within a larger watershed were monitoring is also being conducted to measure loads being discharged from whole Green Branch sub- watershed.

This project has finished the calibration, or before phase (1994-1998), of the project and began the treatment phase in the fall of 1998 in the study watershed. The treatment phase consists of aggressive promotion of nutrient management, the elimination of manure as a nutrient source and cover crops on all available acres in the treatment watershed. These paired watershed results, in conjunction with the models described above, will also provide the means to estimate the overall effectiveness of the watershed pollution control efforts that are initiated to correct the identified problems.

The work identifying pollutant source pathways and timing of the delivery of pollutants has been completed. The investigation will report on nutrient concentrations in shallow groundwater, ground water flow paths and the apparent age of ground water being discharged from the paired watersheds in the fall of 2001. The determination of sediment transport rates and nutrient reservoirs in sediment will also continue through 2001.

B. Characterization of pollutant sources and estimation of pollutant loadings.

1. Review of permit compliance records, inspection and sampling of regulated pollution sources as required to document the characteristics of regulated discharges in the watershed.

Field. Significant sources will be sampled in conjunction with periodic intensive surveys of point and nonpoint sources in the watershed.

Laboratory: Standard laboratory methods will be used for conventional pollutants. Toxic contaminants will be analyzed using methods described in the associated Toxicant Component Work plan.

Data Management: Data will be assembled and verified using appropriate data base software.

Data Analysis and Reporting: Average annual discharge flows and loads will be computed from available sampling data. A report will be prepared listing each source and summarizing its compliance record and pollutant loading history.

2. Sampling of water quality and measurement of streamflow during high and low flow periods at sites located throughout the lower Eastern Shore.

Field: A sub-set of 20 sites has been selected from the larger suite of TMDL sampling sites to cover the major tributaries and source areas These sites will be sampled at least 3 times over the course of the year to characterize a range of flow conditions. Estimates of stream flow will be made at the time of each sampling event.

Laboratory: Standard laboratory methods will be used for conventional pollutants. Toxic contaminants will be analyzed using methods described in the associated Toxicant Component Work plan.

Data Management: Data will be assembled and verified using appropriate data base software.

Data Analysis and Reporting: Instantaneous yields of pollutants will be calculated using discharge and concentration data. Data will be plotted compared between sites to provide an overview of the relative magnitude and range of the pollutant loads from different sources and areas in the watershed. Data will also be used in the calibration of the watershed models described below.

3. Nutrient Loading Assessment

Field: Monitoring sites have been established at Willards, on the Pocomoke River, at the outlet of Nassawango Creek, and on Green Branch, just above the confluence with the Pocomoke River, for continuous discharge and water quality monitoring. Conducted by cooperatively by USGS and DNR, water quality will be sampled biweekly and during storm events.

Laboratory: Standard laboratory methods will be used for conventional pollutants.

Data Management: Data will be assembled and verified using appropriate data base software.

Data Analysis and Reporting: Data will be used to analyze annual trends in flow and concentration, mass loadings for nutrients and suspended solids, and to provide loading coefficients for specific sub-watersheds within the Pocomoke basin.

4. Compilation and summary of available data on pollutant sources in the watershed, including nutrient and pesticide applications (fertilizer, manure, sludge, etc.), BMP implementation and atmospheric deposition on cropland, urban and residential areas.

Field:

1. MDE will continue to develop GIS data layers for home sites with septic systems, public service areas, and sewage application sites and a data base for application rates in watersheds where TMDL's are being developed.
2. DNR in conjunction with the Worcester Soil Conservation District, MDA, NRCS and the local operators will continue to assemble actual application rates and yields within paired watersheds.
3. Estimates of atmospheric deposition will continue to be made using local rainfall data and estimates of constituent concentrations from a number of sources (DNR, UM, SERC).
4. MDA will continue to collect data by sub-watershed on the number and type's of BMP's constructed and acres under management.

Data Management: Data will be coded and managed in appropriate GIS formats.

Data Analysis and Reporting: Data will be utilized in computer models and summarized in tables and/or graphical form as appropriate. Data will also be forwarded to EPA Chesapeake Bay Program for use in the Bay Watershed Model.

5. Development of computer models of the watershed, river and estuary.

Data Analysis and Reporting: Data and information compiled as described above will be used to support the development of computer models of the watershed, stream/river network and estuary. Models are necessary to extrapolate the available monitoring data and information on watershed characteristics to estimate the pollutant load from different sources and areas of the watershed. This information will be used to guide pollution control efforts by helping to set appropriate permit limits and to target voluntary pollution control measures to areas that contain the most significant pollution sources.

The watershed will be modeled using the Hydrologic Simulation Program Fortran (HSPF). Modeling in the subject watersheds will build upon ongoing work by the Chesapeake Bay Program and Maryland's TMDL Program. All significant sources will be included (point, nonpoint and atmospheric). The watershed model will be calibrated to the water quality data collected as described above.

The estuary will be modeled using the Water Quality Analysis Simulation Program (WASP). The estuarine modeling will be used to estimate the relative contributions of point sources and nonpoint sources from the major tributaries that discharge to tidal waters in order to set appropriate discharge limits that will achieve water quality goals. This model will also be required in order to estimate the relative importance of dilution vs. uptake of nutrients by algae and the marshes bordering the estuary.


B. Estimation of effectiveness of management practices

1. Paired Watershed Study

Field: DNR has ongoing work on Green Branch in the Upper Pocomoke. The project is a nested paired watershed study looking at the effect of an aggressive conservation program on nutrient concentrations and loads being discharged from the Green Branch sub- watershed into the Pocomoke River. The paired watershed study is evaluating the effects of the nutrient management BMP's on in stream nutrient concentrations and loads. The treatment phase will consist of aggressive promotion of nutrient management, the elimination of manure as a nutrient source and cover crops on all available acres in the treatment watershed. The paired watersheds are nested within a larger watershed were monitoring is also being conducted to measure loads being discharged from whole Green Branch sub-watershed.

This project has finished the calibration, or before phase (1994-1998) of the project and began the treatment phase in the fall of 1998 in the study watershed.

Laboratory: Standard laboratory methods will be used for conventional pollutants.

Data Management: Data will be assembled and verified using appropriate data base software.

Data Analysis and Reporting: Analysis of the relationship of the nutrient concentrations for the treatment verses control watersheds for the calibration and treatment periods will be performed. Changes in the predicted treatment watershed values will be used to determine the effect of the BMP implementation. The data will also be used to provide nutrient load and yield data for Section B of this work plan.

C. Determine transport pathways and estimate "lag time" of pollutant delivery

1. Determine "lag time" of nutrients in the ground-water.

Need to determine the flow system, associated nutrient concentrations, and ground-water ages in selected areas to determine importance as nutrient delivery pathway. Work for this task has been funded through a coop study between the USGS MD District and the State of MD.

Data Analysis and Reporting: USGS will analyze and interpret ground-water age and nutrient data from Summer, 1998 through the Summer of 2000 sample collection. Results will be summarized in a report to be published this fall (2001). Note: only one lab in the USA does GW age dating so results can take up to six months.

2. Assess deposition and transport of sediments and associated nutrients in the surface water system.

The time of travel sediment and associated P needs to be estimated to understand the relation between sources and delivery. Funding to begin this work will be under the USGS National Research Program and Ecosystem Program efforts. The scope of work (number of sites and analysis) needs to be enhanced with additional State and USGS funds.

Field: USGS will continue to collect data on sediment deposition rates and associated nutrient concentrations in forested wetlands in the Pocomoke river watershed. Sites will consist of transects with clay pads to document sediment deposition and associated nutrient concentrations. (Winter-Spring, 1998 and continuous for at least two years).

Laboratory: Standard USGS laboratory methods will be used for sediment and nutrient
analysis.

Data Management: Data will be assembled and verified using appropriate data base software.

Data Analysis and Reporting: The data interpretation and analysis of data will be conducted during FY99and FY2000.

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