Large-Scale Restoration of Eelgrass (Zostera marina) in the Patuxent and Potomac Rivers, Maryland

Submitted by: Kathryn Busch Rebecca Raves Golden

Maryland Department of Natural Resources Tawes State Office Building, D-2 580 Taylor Avenue Annapolis, MD 21401

March 31, 2009

Final Report to National Oceanic and Atmospheric Administration Grant Award Number: NA03NMF4570470 Project Officer: Peter Bergstrom

Introduction Submerged aquatic vegetation (SAV) in the Chesapeake Bay has experienced several dramatic population declines beginning in the 1930s with the decline of Zostera marina (eelgrass) (Orth & Moore 1984). During the 1960s and 1970s, all species declined baywide coincident with regional water quality degradation and Hurricane Agnes in 1972 (Kemp et al. 1983; Orth & Moore 1983a; Orth & Moore 1984). While tidal freshwater SAV populations have recovered substantially, mesohaline reaches of the lower bay have not recovered (Orth et al. 2008). Numerous areas in the bay, the Patuxent and Potomac Rivers in particular, were densely vegetated with eelgrass. However, distribution is now restricted to Tangier Sound on the lower Eastern Shore of Maryland, a geographic shift of over 50 miles since the 1970s (Orth et al. 2008). Because of the functions SAV serve in maintaining a healthy estuarine ecosystem, SAV restoration has become an important component of Chesapeake Bay restoration. The Chesapeake Bay Program created a Strategy to Accelerate the Protection and Restoration of Submerged Aquatic Vegetation in the Chesapeake Bay. The Strategy, the result of more than a year-long effort among Chesapeake Bay SAV researchers and managers, identifies a variety of actions necessary to increase SAV populations in the Bay. The actions fall into four major categories: 1. improve water clarity sufficient for supporting healthy SAV populations, 2. protect existing beds from impacts by anthroprogenic sources and exotic species, 3. plant or reseed 1,000 acres in strategic locations by December, 2008, and 4. conduct applied research and public education / outreach on the benefits of healthy SAV beds.

When the Chesapeake Bay Program and its partners created the Strategy, attention was given to the need for SAV restoration on a large-scale and eelgrass was identified in the Strategy as one of two species with great potential for large-scale restoration in the Chesapeake Bay. Early planting and reseeding efforts in Maryland and Virginia demonstrated the potential for using eelgrass in restoration projects, but development and refinement of large-scale restoration techniques was necessary. Early eelgrass restoration efforts involved manually transplanting whole adult eelgrass plants in the form of sods, cores or bareroot plants from healthy source beds to restoration locations (Davis & Short 1997; Fonseca et al. 1982; 1994; Orth et al. 1999). Limitations of these methods include the availability and location of donor beds, impacts of harvesting on the donor beds and expense due to the labor and time intensive nature. Attempts have been made to automate this method by utilizing a mechanized planting boat and underwater harvesting and planting machines to accommodate large-scale projects (Fishman et al. 2004; Paling et al. 2001a; Paling et al 2001b). While transplanting adult plants is still utilized as a restoration method, there has been increasing evidence that eelgrass seed dispersal can be a viable option for largescale restoration projects (Granger et al. 2002; Harwell & Orth 1999; Orth et al. 1994; 2003). Seed broadcasting appears to be a more efficient and cost effective technique for SAV restoration (Orth et al. 2000) with the added benefit of not having to remove adult plants from healthy eelgrass beds. As of fall 2005, the Patuxent and Potomac Rivers were two of a few sites in Maryland and Virginia that had undergone the two-year site selection process (test

plantings and water quality monitoring) outlined as a requirement of the strategy for large scale restoration locations. Prior to the decline of SAV beds in Chesapeake Bay between the 1960s and 1970s, the Patuxent River supported populations of SAV including Zannichellia palistris, Ruppia maritima, and Potamogeton perfoliatus (Brush and Davis 1984). Both stratigraphic records and groundtruthing evidence suggests the presence of Z. marina, eelgrass, historically throughout the mesohaline portion of the Patuxent and Potomac Rivers (Pfitzenmeyer & Drobeck 1963; Haramis & Carter 1983; Orth & Moore 1983a; Brush & Davis 1984; Brush & Hilgartner 2000). A resurgence of SAV in the tidal freshwater reach and oligohaline portion of the Patuxent River since 1993 has been attributed to significant reductions in pollutant loads and resulting improvements in water clarity. When this project began, the 2004 aerial survey recorded 220 acres of SAV in the tidal fresh portion, 107% of the 205-acre goal for this portion of the river, and 106 acres in the oligohaline region, 92% of the 115-acre goal for that area (Maryland Department of Natural Resources 2005). However, SAV populations remain sparse in the mesohaline region of the Patuxent River. Only 42 acres were mapped in 2004, far below the 1,634 acre goal for this portion of the river (Maryland Department of Natural Resources 2005). In 2004, SAV coverage in the Potomac River had increased somewhat since 1984 but acreage was still far short of acreage goals in some regions. The 2004 aerial survey recorded 1,256 acres of SAV in the tidal fresh portion, 59% of the 2,142-acre goal (Maryland Department of Natural Resources 2005). In the oligohaline region 1,408 acres were mapped, exceeding the 1387-acre goal (Maryland Department of Natural Resources 2005). SAV populations in the mesohaline region covered 3,063 acres, only 43% of the

7,088-acre goal for that portion of the river (Maryland Department of Natural Resources 2005). In this multi-year investigation, we conducted and examined outcomes of largescale eelgrass seed restoration efforts in two Maryland river systems from 2004 to 2008. Multiple seed collection, processing, storage and seed dispersal techniques were designed and compared. The efficiency of manual eelgrass seed collection (snorkeling and SCUBA) was compared to mechanical harvest. Several seed storage conditions and processing techniques were investigated in order to maximize viable seed yield for fall seed broadcast activities. The associated costs for two seed dispersal techniques, fall seed broadcast and spring seed bag methods, were also compared. In addition to methods development and refinement, this investigation analyzes the influence of site-specific SAV habitat conditions on eelgrass seedling establishment and long-term survival, as well as evaluates the relative success of this multi-year large-scale eelgrass restoration project.

Methods Study Sites Locations for large-scale restoration activity were determined using a geographical information system (GIS) based SAV Restoration Targeting System (Parham & Karrh 1998). The model incorporates seven layers of habitat data (light attenuation, seston, chlorophyll a, dissolved inorganic nitrogen and phosphorous, bathymetry and salinity) to evaluate the potential of a particular area to support SAV populations. Habitat data from three years (2000-2003) prior to the start of the project was incorporated into the model and updated in subsequent years of the project as data became available. Five sites in the lower mesohaline Patuxent River and five sites in the lower mesohaline Potomac River, MD were identified as potential habitats for eelgrass recolonization based on the results of Maryland DNRs SAV Restoration Targeting System. The following sites were identified on the Patuxent River; Parrans Hollow (lat 38.4119N, long 76.5275W), Jefferson Patterson Park (lat 38.4073N, long 76.5211W), Myrtle Point (lat 38.3293N, long 76.4916W), Hungerford Creek (lat 38.3496N, long 76.4720W), and Solomons Island (lat 38.3150N, long 76.4542W) (Figure 1). Five sites on the Potomac were also identified; Cherryfield Point (lat 38.1303N, long 76.4596W), Piney Point (lat 38.1380N, long 76.5027W), Sage Point (lat 38.1167N, long 76.4333W), St. George Island (lat 38.1333N, long 76.4833W), and Kitts Point (lat 38.1105N, long 76.4245W) (Figure 2). The selected sites were shoal areas which generally followed the 0.5-1 meter mean low water (MLW) contour and historically supported eelgrass populations.

Adult plant collection and transplanting Adult eelgrass transplants were established to ensure that areas identified by the site selection model (Parham & Karrh 1998) were capable of supporting eelgrass survival and growth, as well as to provide reference data for seedling establishment. Adult eelgrass plants were collected from donor beds in Tangier Sound in 2004, and Chincoteague Bay in 2004, 2005 and 2006. Eelgrass plants were harvested with shovels and gently sieved to remove sediments (Orth et al. 1999), placed in coolers and transported to the PPAF where they were held in indoor tanks with metal halide grow lights (70-200 mol photon m-2 sec-1) and flowing estuarine water until transplanted (< 1 week). A small portion of the total eelgrass plants used in 2004 and 2005 was grown from seed in culture at the PPAF. Planting units (PU), each consisting of two shoots with intact rhizomes and roots, were anchored to the bottom with bent bamboo skewer staples (Davis & Short 1997). Planting units were assembled at the transplant site and planted at a density of 64 PU/m2 in three replicate 1m2 plots adjacent to seed dispersal areas.

Eelgrass Seed Collection To determine the progress of eelgrass seed development and maturation, surveys of reproductive shoot and seed development within potential donor beds in the Chesapeake Bays Tangier Sound (38 00.530 N 75 58.349 W), the Little Annemessex River (37 58.479 N 75 52.255 W), the Big Annemessex River (38 01.718 N 75 50.632 W) and Sinepuxent Bay (38 24.843 N 75 15.085 W) in Marylands Coastal Bays system began in March and continued through April and May (2003-2007) (Figure 3). A small number of reproductive spadices were removed from eelgrass plants and the mean viable seed yield per reproductive spadix was estimated. Large-scale collection did

not begin until at least 50% of the seeds within reproductive shoot spadices were mature in order to ensure harvesting occurred during the peak of seed production. Reproductive shoots were collected manually using SCUBA (Granger et al. 2002) or snorkeling in 2003 and 2006. Between 2004 and 2008, a mechanical harvest machine (Pristine Marine, M J McCook & Associates, La Plata, Maryland) was utilized (Figure 4). Immediately following collection, eelgrass reproductive material was manually loaded into nylon mesh bags (113.5 L), secured at a nearby dock, and kept submerged in ambient water until utilized for one of two seed dispersal methods.

Large-Scale Seed Dispersal Methods Seeds were dispersed in large-scale plots, from 0.1 to 5 acres, at various sites on the Patuxent and Potomac Rivers between 2003 and 2008 utilizing two methods. A portion of the seed material collected was transported to the Piney Point Aquaculture Facility (located in St. Marys County, Maryland) by boat or truck within 24 hours of collection where the material underwent processing and storage procedures in order to extract mature seeds for fall seed broadcast. The remainder of the harvested material was transferred to seed bags for immediate spring deployment.

Fall Seed Broadcast: Processing, Storage and Broadcast After collection, seed material was transferred from mesh bags into one of eight, 75,708 L (9.8 x 9.8 x 10.4-m) or one of sixteen 37,097 L (6.1 x 6.1 x 1.2-m) greenhouse basins located at the Piney Point Aquaculture Facility. Configured as an open system, each basin received a constant inflow of filtered water from nearby St. George Creek (~10-18psu) to allow for daily water replacement. In order to maintain similar salinity

levels to the seed collection areas (~14psu), each basin was augmented with aquaculturegrade sea salts as necessary. For the first 3 years of the project (2003-2005) 208-L drums filled with salt were placed directly below the water inlet in each individual basin to rapidly dissolve the salt and increase salinity above ambient levels. From 2006 to 2008, salinity was closely controlled using a concentrated brine solution added to the main incoming water line feeding all basins. In addition, each basin was aerated with evenly positioned air stones at a density of one per 1,700 L and maintained 5-6 mg/L dissolved oxygen levels. While in the basins, the eelgrass seeds slowly separated from the decomposing reproductive shoots over the following 3 or 4 weeks. After all the seeds had been released and settled to the bottom of the basins, the seed/reproductive shoot slurry was pumped by a diaphragm pump into a series of stacked settling trays to allow the passive accumulation of seeds while discarding the non-seed material. The seeds that were separated from the bulk of the vegetative material were then transported to another filter system and moved through a series of progressively smaller filters (2380 m, 1800 m and 1000 m, respectively) to remove non-seed material. Once separated from all other reproductive material, the seeds were held in a 9,464 L tank recirculating system. System water was aerated, held at ambient temperature (18-28oC) and kept at 14psu until dispersal. Beginning in 2006, seeds were stored in the same recirculating system however, salinity was increased to 18psu and water temperature was held at a constant 18oC. Bubble aeration in the seawater trays was replaced with aeration in the head tank, with each seawater tray equipped with a laminar flow system. Seed densities were estimated and viability determined by counting

replicate 5 ml samples of seed material. Viability was determined using a squeeze test (R. Orth 2004, Virginia Institute of Marine Science, Gloucester Pt., VA, personal communication). Eelgrass seeds were broadcast manually (Orth et al. 1994) during the fall of 2003, 2006, 2007 and 2008. A mechanical seed sprayer (C& K Lord, Inc) was utilized to broadcast seeds in the fall of 2004 and 2005. The seed sprayer was mounted to a boat, capable of evenly dispersing seeds at suitable densities (100,000 to 300,000 seeds/acre) at the rate of 10 minutes/acre (Figure 5). The flow of the seed sprayer was calibrated and adjusted to distribute seeds uniformly at the desired density. Seeds were loaded into the seed broadcast machine where the seeds were mixed with water and expelled into the water column. All seed broadcasts took place in October before the ambient water temperatures dropped below 15C, prior to eelgrass seed germination (Moore et al. 1993; Orth & Moore 1983b). Spring Seed Bag Method A portion of collected eelgrass reproductive seed material was prepared for immediate deployment following a buoy-deployed seeding system (BuDSS) developed by Pickerell et al. (2005; 2006) with modifications. A known volume of reproductive material was subsampled and the seeds enumerated. Based on the seed estimates, a volume of reproductive seed material necessary to achieve the desired seeding density was transferred to pre-measured, coarse (7 x 7-mm) mesh bags with buoys and attached to 1.8-m polypropylene line and fastened securely with cable ties to cinderblocks (0.1 x 0.2 x 0.4-m) (Figure 6). Completed seed bags were transported to restoration locations and deployed in a grid pattern ranging from 6 x 5 bags to 11 x 11 bags spaced 10 meters

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apart. The goal seeding density at each location was approximately 37 seeds/m2 despite varied plot size at each location.

Eelgrass Monitoring and Analysis Adult plant monitoring Adult eelgrass transplants were monitored for change in density one month, six months and twelve months after transplant. In 2005 and 2006, additional monitoring was performed monthly throughout the summer (May-August). If planting units were observed twelve months after sampling, monitoring continued during the spring, summer and fall of subsequent years. Transplant survival was calculated as the proportion of planting units observed during each monitoring period divided by the number of initial transplants. When individual planting units in each plot could not be distinguished due to lateral expansion, survival for that plot was assumed to be 100%. Seed plot monitoring Seedling establishment was calculated as the proportion of initial seeds dispersed that were observed as germinated seedlings the following spring. Eelgrass shoot density was monitored along two to four non-destructive, 1 m2 belt transects (Burdick & Kendrick 2001) in each of the seed dispersal plots. Initial eelgrass monitoring occurred in the spring after seed dispersal with successive monitoring in the summer (three months after initial monitoring) and fall (six months after initial monitoring). The survival of plants in seed dispersal plots was calculated as the proportion of initially established seedlings that were observed in the fall. Eelgrass density was also monitored in the

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spring, summer and fall of subsequent years of the project and long-term survival was assessed at the end of the five year project. Analyses were performed using the statistical software package SAS version 9.1 (SAS Institute Incorporated, Cary, NC, U.S.A.). Due to the unbalanced nature of the data, one factor Welchs weighted analysis of variances (ANOVAs) were performed to assess treatment effect (Seed dispersal method, Dispersal year, River and Site) on seedling establishment and eelgrass survival data. Plant density data was also compared for each treatment effect (Dispersal year, Seed dispersal method, River and Site nested in River) using repeated measures ANOVAs (PROC MIXED in SAS 9.1 using an autoregressive covariance structure). Following significant interactions with time, univariate ANOVAs were used to compare Seed dispersal method and Dispersal year at each time period. For adult plant transplant survival data, repeated measures ANOVAs (PROC MIXED in SAS 9.1 using an autoregressive covariance structure) were performed to evaluate treatment effect (Year, River and Site). Following significant interactions with time, River and Site were compared at each time using univariate ANOVAs. Homogeneity of variance was assessed using Levenes test. Following a significant ANOVA, Tukeys or Dunns tests for post hoc multiple comparisons were performed.

Habitat Monitoring and Statistical Analyses Spatially intensive water quality monitoring was conducted monthly during the eelgrass growing season (March - November) throughout the lower mesohaline portion of the Patuxent River from 2004 to 2006 and the lower mesohaline Potomac River (St. Marys River portion) from 2004 to 2007. Georeferenced surface water temperature,

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conductivity, pH, dissolved oxygen, turbidity and fluorescence data were collected every four seconds by a shipboard YSI 6600 sonde (Yellow Springs, OH) (Michael et al. 2008). Two YSI 6600 EDS sondes on the Patuxent River and three sondes on the Potomac River were deployed during the monitoring period (2004-2007) to provide temporally intensive habitat assessments of adjacent restoration study sites throughout the SAV growing season (April-October). On the Patuxent River, stations were deployed at the Chesapeake Biological Laboratory (lat 38.3167N, long 76.4526W) in 2004 and 2005 and Pin Oak (lat 38.4088N, long 76.5218W) in 2004 through 2007. On the Potomac River, stations were deployed at Piney Point (lat 38.1377N, long 76.5058W) from 2004 to 2007, near Sage Point (lat 38.1135N, long 76.4285W) in 2004 and 2005 and in St. George Creek (lat 38.1311N, long 76.4934W) in 2006 and 2007. Each sonde collected water temperature, conductivity, pH, dissolved oxygen, turbidity and fluorescence data every 15 minutes 0.5 meters above the bottom (Michael et al. 2008). Non-parametric ANOVAs (Kruskal-Wallis) were performed on temporal water quality data (April-October) to assess differences in locations for each year (2004-2007). The data generated from the sondes and shipboard GPS were mapped in ArcGIS (ESRI, Redlands, CA) for each monthly cruise. The data were then interpolated using the Inverse Distance Weighted method to create a grid with a pixel dimension of 50 meters. Mean grids for each parameter were generated for each river and overlaid with the seed dispersal plot grids to yield water quality conditions directly above the restoration sites. These data were then analyzed using Welchs t-test to determine differences in spatial water quality among seed dispersal plots on the Potomac and Patuxent Rivers.

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Cost analyses To compare the cost effectiveness of each dispersal method, the total cost of the particular method was divided by the total number of viable seeds dispersed using that method for each year of the project (2004-2008). The total cost for seeding one acre was then calculated by multiplying the cost per seed by the specified seeding density (200,000 seeds/acre). When determining the total cost for each method, all costs associated with seed collection, processing, storage and dispersal, such as staff and volunteer labor, harvesting equipment costs and expendable supplies were included. However, these costs do not include additional one-time equipment purchases, utilities, project management or monitoring.

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Results Study Area Five sites on the Patuxent River and five sites on the St. Marys River (Potomac River) were identified as suitable habitats for eelgrass restoration activities between 2003 and 2008. All restoration efforts for this project took place on the Patuxent River between 2004 and 2006. However, due to the lack of success of seeding efforts as well as limited bottom area for seeding, restoration efforts shifted to two of five previously identified restoration locations on the Potomac River, St. George Island and Cherryfield Point from 2006 to 2008. For full comparison, data from all years on both rivers were included in this report for analysis.

Eelgrass Seed Collection Comparison The total volume of eelgrass reproductive material collected from 2003-2008 was 414,803 L. Annual collection rates were highly variable and dependent on collection time and method (Table1). Collection rates ranged from 392 L/day in 2006 to 22,720 L/day in 2005, with a mean annual collection rate of 9,483 L/day. The mean mechanical collection rate (9,529 L/day) was also six times greater than the mean manual collection rate (1,515 L/day).

Eelgrass Seed Processing and Storage The volume of eelgrass seeds processed ranged from 32.5 L in 2006 to 109.8 L in 2005, with a mean annual processing volume of 66.6 L (Table 1). The amount of seeds available for processing and storage was dependent on the amount of reproductive

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material collected, as well as the proportion of eelgrass reproductive material utilized in spring seed bag dispersal and other restoration projects. The mean percentage of viable eelgrass seeds remaining in the fall after processing and storage procedures was 35% with a range of 7-87% (Table 1).

Eelgrass Seed Dispersal Methods Comparison A total of 13,498,000 eelgrass seeds were dispersed from 2003 to 2008, with a mean of 1,687,250 seeds each year of the project (Table 1). The portion of eelgrass seeds dispersed through each method varied by year and was dependent on; 1)amount of reproductive material collected 2)available shoal area for spring seed bag deployment and 3)amount of tank volume at the processing and storage facility. During the six years of this project, one third of the eelgrass seeds were dispersed via the fall broadcast method and the remaining two thirds were dispersed utilizing the spring seed bag method.

Eelgrass Monitoring and Analysis Adult plant monitoring All adult test plantings on the Patuxent River died within one year of planting (Figure 7). Plantings at several sites on the Potomac River survived several years including those planted in at St. George Island in 2004 and 2005 and at Cherryfield Point in 2006. With the exception of a few signs of disruption, such as that of a ray, most adult plantings declined during the summer months, June through August. Adult transplant survival decreased significantly over time (Table 2). Mean observed transplant survival was 73% one month after planting, 58% six months after

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planting and 10% twelve months after transplant. While there were significant differences in adult transplant survival among sites one month and six months after planting, adult transplant survival at twelve months was statistically similar (Tables 2 and 3). Transplant survival was significantly different between rivers twelve months after transplant as no planting units were observed on the Patuxent River (Tables 2 and 3). However, mean transplant survival at twelve months was 26% and 17% at St. George Island and Cherryfield Point, respectively. Seed plot monitoring Eelgrass seeds were dispersed on the Potomac and Patuxent Rivers between 2003 and 2008. All seeding on the Patuxent River and 2006-2008 seeding on the Potomac River were funded through this project. A total of 13,498,000 seeds were dispersed using 2 seeding methods in 41 discrete planting areas at 10 different locations, five on each river (Tables 4 and 5). Eelgrass seedlings were observed in a majority (69%) of the plots during initial monitoring in the spring following seed dispersal. Seedling establishment, or the percentage of seeds observed as seedlings, in restoration plots was highly variable and ranged from 0 to 3.7% depending on site, dispersal method and year (Table 6). Establishment was generally 1.5 times higher in fall seed broadcast plots than in spring seed bag plots and twice as high for areas dispersed in 2007 than those dispersed in previous years (Table 7). While seedling establishment was roughly equivalent for both the Patuxent and Potomac Rivers (Table 7), the number of eelgrass seedlings observed was highly variable among sites. Highest mean establishments at a particular seeding location (1.4%) occurred at St. George Island on the Potomac River and Jefferson

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Patterson Park on the Patuxent River. Several sites (Piney Point and Solomons Island) had no observed seedling establishment regardless of dispersal method. Eelgrass shoot density varied significantly with time, with increases in shoot density observed during early summer monitoring, followed by decreased densities observed in the fall (Figure 8). Shoot density was significantly higher in fall seed dispersal plots than in spring seed bag plots (Table 8, Figure 8A). Density was also significant for dispersal year (Table 8, Figure 8B). Both dispersal method and year had significant interactions with time (Table 8). Univariate ANOVAs showed no significant differences in eelgrass shoot density between dispersal method and year during initial spring monitoring or fall monitoring (Table 9). However, eelgrass densities were significantly different for dispersal method and year during summer monitoring (Table 9, Figure 8A & Figure 8B). Observed eelgrass density generally declined over the course of the first year of monitoring (Tables 7 & 10). Mean plant survival, or the percentage of initial seedlings observed as plants after one year, was higher on the Potomac River (37 60%) than on the Patuxent River (0 0%). Plant survival was generally twice as high in fall seed broadcast plots (36 54%) than in spring seed bag plots (15 49%) and higher in 2006, compared to other dispersal years six months after initial monitoring. Mean long-term survival (plants observed in the fall of 2008 as a percentage of initial seedlings) was also greater on the Potomac (338 750%) than on Patuxent River (0 0%) (Figure 8C) and higher in fall broadcast plots (467 922%) than in spring seed bag plots (42 108%) (Table 7 and Figure 8A). Plots with seeds dispersed in 2006 had greater long-term survival (1519 1622%) than plots dispersed in other years (Table 7).

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Long-term survival was 0% at all sites except for St. George Island and Cherryfield Point on the Potomac River, where the mean percent increase in eelgrass shoot density was 559 and 89%, respectively (Table 11).

Habitat Monitoring and Statistical Analyses Two YSI 6600 EDS sondes on the Patuxent River and three sondes on the Potomac River were deployed during the monitoring period (2004-2007) to provide temporally intensive habitat assessments of adjacent restoration study sites throughout the SAV growing season (April-October). All temporal data (water temperature, salinity, dissolved oxygen, pH, fluorescence and turbidity) collected by the YSI 6600 EDS sondes were highly significant among monitors for each year of the project (Table 12). Temporal data including minimum daily dissolved oxygen, maximum daily water temperature, maximum daily turbidity and maximum daily chlorophyll at each of the five stations are presented in Appendix A and discussed further in the discussion. Cumulative frequency analysis of turbidity, chlorophyll, dissolved oxygen and temperature values was performed and is graphically presented in Appendix B and discussed in the discussion. Spatially intensive water quality monitoring was conducted monthly during the eelgrass growing season (March - November) throughout the lower mesohaline portion of the Patuxent River from 2004 to 2006 and the lower mesohaline Potomac River (St. Marys River portion) from 2004 to 2007. Spatial turbidity data is presented in Appendix C. Results of the GIS analysis indicated that spatially interpolated mean chlorophyll and turbidity were significantly lower (t.05[1] <0.0021) and mean water temperature and

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salinity values were significantly higher (t.05[1] <0.0001) on the Potomac River than on the Patuxent River.

Cost Anaylsis The cost of seeding one acre of suitable bottom varied greatly between the spring and fall dispersal methods. Spring seed bag dispersal costs ranged from $2,702/acre to $39,654/acre between 2004 and 2007 (Table 13). Fall seed dispersal costs were 2 to 15 times greater than spring seed bag dispersal costs, ranging from $17,009 to $67,085 per acre between 2004 and 2008 (Table 13).

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Discussion Harvesting of eelgrass seeds for restoration has traditionally involved hand collection using SCUBA or snorkeling (Granger et al. 2002; Orth et al. 2006a). While this can be effective for small-scale restoration, innovative techniques for enhanced seed collection were needed in order to conduct eelgrass restoration on a larger (multi-acre) scale. Eelgrass reproductive material collection was increased from approximately 22,796 L in 2003 using manual harvesting to approximately 89,918 and 204,482 L in 2004 and 2005, respectively. This was due primarily to the use of a mechanical harvester in 2004 and 2005. The volume of material collected in 2006 decreased greatly due to mass eelgrass mortality in the late summer of 2005 (R. Orth 2005, Virginia Institute of Marine Science, Gloucester Pt., VA, personal communication)., equipment problems and adverse weather conditions. However, mechanical collection volumes and rates improved in 2007 and 2008. While manual collection allowed for a less destructive selection of eelgrass reproductive material, the amount of material and the per man-hour collection rates were unable to in provide enough eelgrass seeds for large-scale restoration. Mechanical harvesting dramatically improved material collection rates and volumes. However, this method was more expensive, logistically more difficult and is less selective in the plant material collected. A greater amount of non-reproductive plant material is collected due to the inefficient design of the harvesters cutting mechanism. Steps were taken to reduce damage to the existing eelgrass donor beds during mechanical collection. The depth of the cutting blades on the harvester was adjusted to only remove the upper third of the eelgrass plants, thus avoiding damage to the rhizome

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mat and lower vegetative parts of the eelgrass plants. Harvesting also took place over a large area (several acres) to assure that sufficient seeds remained for the maintenance of the donor beds. Following mechanical harvest, replicate haphazard 1m2 quadrats were surveyed in harvested and adjacent unharvested areas to compare shoot height and density between donor beds and nearby control beds. No substantial differences in plant height, shoot density, or apparent vigor of the plants themselves existed between the harvested and unharvested beds. In addition, low level (1:24,000) aerial photography taken shortly after seed collection confirmed that the areas that were harvested in May were still densely vegetated (MD DNR unpublished). The lack of eelgrass seed physiology research made storing large numbers of seeds under man-made conditions for 4-6 months very difficult. Seed viability was greatly increased by changes made to storage conditions and procedures over the course of the study. After much trial and error, it was ultimately determined that seeds stored at 18oC and 18-20 psu in filtered water resulted in the highest numbers of viable seeds. The continued research and modification of seed storage protocols led to a drastic increase in seed viability and subsequent increased quantities of seeds available for broadcast. This dramatically affected the costs associated with this method over the years. While fall seed broadcasting was more expensive, there are several advantages to this method. Because eelgrass seeds are negatively buoyant they settle to the sediment surface where they are rapidly incorporated and generally do not move far from where they settle (Orth et al. 1994). Germination begins in mid-October when water temperatures drop below 15oC (Moore et al. 1993), therefore seeds dispersed in the fall

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germinate shortly after being dispersed. Since seeds germinate soon after dispersal, seed predation, removal from the system through transport to unsuitable habitat areas and deep burial are less likely. Seeds broadcast in the fall require less manpower than those dispersed in the spring. Fall seed broadcast was simplified in 2006 using a manual seed broadcasting system as we realized that the calibration of the mechanical seed sprayer took more time than it saved. A buoy-deployed seeding system (BuDSS) developed by Pickerell et al. (2005; 2006) was modified slightly and used as an alternative method to broadcast seeding in the fall. Immediate deployment of reproductive material in the spring eliminates summer seed storage, reducing the number of seeds lost to processing and decreasing the expense and labor requirements associated with seed transport, processing, and storage. Alleviating long-term seed storage can be a significant advantage if the infrastructure is not present to house a seed storage operation thus reducing major capital investment and therefore project costs. There are several limitations to the spring seed bag dispersal method. While this method mimics the floating and rafting of reproductive shoots during the natural phenological schedule (Pickerel et al. 2005), seeds dispersed in the spring will remain in the sediment for 4-5 months before germinating in mid-October when water temperatures drop below 15oC (Moore et al. 1993). During this time, seeds are susceptible to predation, deep burial, or transport out of the suitable growing area. Some species feed directly on eelgrass seeds and can affect seed germination rates (Fishman & Orth 1996). Deep burial of seeds below the redox potential discontinuity prevents the developing plant from receiving light which may be crucial to germination (Bigley 1981). This

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method is extremely labor intensive requiring the deployment and retrieval of large numbers of seed bag units from seeding areas. During the 3 week period when the seed bag units are deployed, they can create a navigational hazard in the waterway. Vandalism, though not documented, can also be a concern. Both collection methods were highly dependent on weather conditions and eelgrass physiology (reproductive shoots length, reproductive shoot density) during the collection period which ultimately affected the seed yield and associated costs. Operating costs for seed collection, processing, storage and dispersal were fairly similar each year, so total costs were inversely proportionate to the number of eelgrass seeds collected. Therefore in years (2006, 2007) with lower collection yields due to poor reproductive success of donor beds or adverse weather conditions, costs per viable seed (and subsequent cost per acre) were much higher. Fall seed broadcast dispersal was consistently more expensive than spring seed bag dispersal due to the costs associated with eelgrass seed processing and storage. The high relative costs are due to the maintenance of adequate seed processing and storage infrastructure. Specifically, large quantities of aquaculture grade sea salt and electricity to run pumps, aerators, and heat pumps to process seeds and maintain optimum storage conditions, as well as the large amount of labor required to staff such a facility, were the primary costs. Similar studies comparing these two methods in the Chesapeake Bay found the fall broadcast was up to 17 times more expensive than seed bag deployment (varying seeding densities) (Shafer & Bergstrom 2008). In addition to higher operating costs for the fall method, low seed viability after summer storage increased the cost per viable seed and therefore the cost per acre. In

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2004, only 7% of seeds collected were available for dispersal leading to significantly higher costs than in 2005 when a larger seed harvest combined with 20% seed viability resulted in much lower costs. Despite increased seed viabilities in 2006 and 2007 (87 and 21%, respectively), less significant seed harvest and a significant investment in supplies to refine the seed processing and storage process resulted in much higher fall costs. In 2008, a similar number of seeds were harvested as in 2007, however, a 60% seed viability after storage coupled with a decrease in supply costs to store seeds for the summer resulted in a significant decrease in costs. Overall, the disparity in the cost per seed was due to a combination of variations in the seed yield from collection and seed viability after summer storage. In years with abundant seed harvest (>10 million seeds) and optimal seed viability after summer storage (>20%), large-scale eelgrass seeding costs can occur at reasonable costs ($2,702/acre, spring seed bags and $17,009/acre, fall seed broadcast). Observed eelgrass seedling establishment was highly variable, ranging from 0 to 3.7% of seeds dispersed. Germination rates in this study fell within range of previously reported seedling establishment for natural eelgrass populations (< 10%) (Harrison 1993) and in eelgrass seed restoration studies in the Chesapeake Bay region (0.6-39.8%) (Orth et al. 1994, 2003, 2006a, 2006b, 2008b; Harwell & Orth 1999; Orth & Marion 2007). Eelgrass seedling establishments ranging from 5 to 15% have been reported for the Delmarva Coastal Bays, VA (Orth et al. 2006b), the Peconic Estuary, NY (Pickerell et al. 2006) and Rhode Island (Granger et al. 2002). Of the 36 seed dispersal plots, 13 had no observed seedling establishment. The number of plots with no observed seedling establishment declined throughout later years

25

of the project as seed dispersal efforts were concentrated in sites with previous seedling establishment and survival. This suggests that site selection is a vital component of eelgrass restoration as seedlings were never observed at two sites (Piney Point and Solomons Island), others sites (Cherryfield Point, Hungerford Creek, Myrtle Point, Parrans Hollow, Sage Point and Kitts Point) had mixed success and two sites (St. George Island and Jefferson Patterson Park) consistently had eelgrass seedlings establish. While the spring seed bag method had a greater number of plots with seedling germination, the mean establishment percentage of those plots was two times less than in plots dispersed with seeds in the fall. While not statistically significant, the differences in seedling establishment between seed dispersal methods suggest that eelgrass seeds dispersed in the spring may be more susceptible to biotic and abiotic conditions prior to germination. Eelgrass seeds utilized in the spring seed bag method are dispersed in late May or early June and can remain in the sediment for up to six months before environmental conditions (water temperature and sediment redox potential) cue germination (Moore et al. 1993). Seeds are susceptible to predation (Wigand & Churchill 1988; Fishman & Orth 1996), entrapment by benthic invertebrates (Luckenbach & Orth 1999), deep burial (Bigley 1981; Churchill 1992), or transport out of the suitable growing area due to near-bottom currents (Orth et al. 1994; Harwell & Orth 1999) before the required conditions for germination are met. However, eelgrass in spring seed bag plots persisted for as long as plants in fall seed broadcast plots, regardless of relative seedling establishment. Of the nine plots that had eelgrass survive for one year, three were dispersed by the spring bag method. Fifteen plots had eelgrass persist longer than two years, and two-thirds were the result of spring

26

seed bag deployments. Eelgrass at sites with average seedling establishment also persisted for roughly the same, in some cases longer, periods of time when compared to sites with relatively high seedling establishment. These observations suggest that eelgrass persistence in restoration plots is not solely dependent on initial seedling establishment or seed dispersal method. Mean observed eelgrass shoot density was higher during the summer (July) than in the fall (October). Shoot densities increased from the spring (April) monitoring to summer (July) monitoring period, and then declined to below-spring densities in the fall (October). The fluctuation of plant density over time was typical of eelgrass populations in the Chesapeake Bay. Orth & Moore (1986) reported maximum eelgrass shoot densities in June and July, minimum values in September following a summer defoliation and the emergence of new shoots in October with shoot production continuing through the winter and spring. Light limitation and temperature stress are thought to be the main contributors to the late summer reductions in eelgrass shoot density (Evans et al. 1986; Orth & Moore 1986; Moore et al. 1996). Many of the seedlings were also clumped in groups and field observations indicated that eelgrass plants were interspersed with areas of bare bottom. Therefore, our reported mean plant densities include areas of higher density plants and unvegetated areas. Observations from other studies have also reported similar seedling clumping (Orth et al. 2003, 2008b). While densities of eelgrass in our restoration plots were extremely low compared to eelgrass shoot densities observed in established natural beds (Orth & Moore 1986; Maryland Department of Natural Resources, unpublished data),

27

shoot densities were comparable to eelgrass restoration areas in the lower York River, VA (Orth & Marion 2007). The consistent loss of eelgrass in restoration sites during the summer (JulyAugust) suggests that this is a critical time period for eelgrass survival. While eelgrass was not observed in some plots the first summer and fall monitoring periods, plants were seen in these areas during spring and summer monitoring in subsequent years. Regardless of year, defoliation occurred during the summer (July-August). Additionally, a summer die-off of eelgrass in summer of 2005 (Moore & Jarvis 2008) caused baywide declines in natural populations, and the impact of this die-off can be seen in our monitoring results. Adult transplants planted in the fall of 2004 only survived the 2005 summer conditions at St. George Island. Eelgrass plants grown from seeds dispersed in 2005 (germinated after summer die-off) were 38 times denser than plants established in 2004 (germinated prior to summer die-off), suggesting that the 2005 summer conditions had major impacts on plant densities in our restoration sites. Our results demonstrate that adult transplant survival is indicative of the survival of eelgrass grown from seed in restoration areas. Sites where transplants persisted longer than 12 months also had long-term survival of eelgrass plants grown from seed. The percentage of surviving transplants declined over time and only two sites on the Potomac River had transplants survive past one year. Eelgrass plants transplanted in 2005 and 2006 at Cherryfield Point persisted 20 (33% survival) and 19 months (10% survival), respectively. Eelgrass transplanted in 2006 at St. George Island had 16% survival at 19 months and when observed the next month, planting units had coalesced and covered the entire planting area. Transplants from earlier years, 2004 and 2005, at the St. George

28

Island site persisted 31 months, with survival of 1.5% and 33%, respectively. No planting units were observed during the following fall monitoring period (24 or 36 months after transplant) for either location. Additional monthly monitoring during the summer of 2005 and 2006 (May-September) revealed that transplant density decreased drastically between July and August and above-ground biomass was absent by September or October, indicating that the summer conditions affecting seedling survival also impact adult transplant survival as well. The lack of long-term survival of eelgrass on the Patuxent River and the consistent defoliation of eelgrass during the summer suggests that eelgrass survival is dependent on site-and season-specific environmental conditions not utilized in the initial restoration site selection process. Water temperatures greater than 25oC (Rasmussen 1977; Nejrup & Pedersen 2008) can stress eelgrass, and when temperatures exceed 30oC, eelgrass die-backs have been reported (Orth & Moore 1986; Moore & Jarvis 2008). High water temperatures can disrupt eelgrass photosynthetic and metabolic processes (Evans et al. 1986; Marsh et al. 1986; Zimmerman et al. 1989; Nejrup & Pedersen 2008). High temperatures and decreased photosynthetic rates also affect the internal oxygen balance of eelgrass (Greve et al. 2003), leading to sulfide intrusion of the rhizome and meristematic tissues (Pederson et al. 2004) and increases in anaerobic metabolites (Pregnall et al. 1984; Smith et al. 1988), ultimately affecting eelgrass photosynthetic rates, growth and survival (Goodman et al. 1993; Holmer & Bondgaard 2001). Several recent summertime eelgrass mortalities have been associated with low oxygen concentrations and high sulfide levels (Holmer & Bondgaard 2001; Greve et al. 2003, 2005; Plus et al. 2003).

29

It is possible that similar stressful conditions (high temperatures and low dissolved oxygen) contributed to the observed summer eelgrass declines in our restoration sites. Summer (July-August) water temperatures exceeded 30oC more frequently on the Patuxent River in all years of the project. Annual differences in the frequency of extreme water quality conditions were also evident during the four years of the project, particularly during the summer of 2005. On the Patuxent River, water temperatures exceeded 30oC 27% of the time during July and August of 2005, with a maximum temperature of 35C. Water temperatures reached 32C in the summer of 2005 on the Potomac River and exceeded 30C 22% of the time. Dissolved oxygen concentrations below 2.5 mg/L were observed 2% of the time on both rivers during the summer of 2005. The frequency of high temperature events was greatly reduced in all other years of the project. Frequent drops in dissolved oxygen were measured on the Potomac River in 2006, however, these observations were collected from one monitor adjacent to a restoration site with similar summer eelgrass declines, suggesting that periods of low summer dissolved oxygen affected eelgrass survival. Moore and Jarvis (2008) reported similar vegetative eelgrass die-offs and increases in the frequency of extreme environmental stressors during the same timeframe of our project. Concurrent losses of eelgrass in our restoration plots suggest that even short-term exposure to the combination of multiple stressors can severely hinder eelgrass restoration, regardless of previous restoration success. Our results indicate that seed dispersal can be an effective method for large-scale eelgrass restoration in the Chesapeake Bay. Successful techniques for large-scale eelgrass seed collection, storage and dispersal have been designed and implemented. We

30

were able to determine the most efficient seed collection method, refine the summer seed storage techniques to increase seed viability and determine the approximate costs associated with large-scale restoration utilizing eelgrass seed. Models to strategically target potential areas for large-scale eelgrass restoration had only moderate success in predicting conditions necessary for restored eelgrass growth and survival. This study revealed flaws in utilizing only water quality in restoration site selection, given the high variability in observed eelgrass seedling establishment and survival at adjacent sites with similar water quality. While seedling establishment and survival are highly variable and dependent on restoration site and environmental conditions, eelgrass established from broadcast seeds has persisted at some sites for up to four years. However, more research on the tolerances of restored eelgrass to environmental stressors is warranted.

Recommendations for future restoration: The use of seeds is a practical option for large-scale Z. marina restoration in the Chesapeake Bay. When practical, mechanical collection of Z. marina seed should be used as it is more efficient than manual collection using snorkeling/SCUBA and can be accomplished without significant harm to donor beds. Z. marina seeds should be processed and stored at 18oC and 18-20psu in filtered water to yield the highest numbers of viable seeds. The cost for seeding one acre of bottom with Z. marina seeds ranged from $2,700 to $39,600 using the spring seed bag method and from $17,000 to $67,000 using the fall seed broadcast method. The disparity in costs per seed was due to a combination of variations in the seed yield from collection and seed viability after summer storage. Fall seed broadcast resulted in greater Z. marina seedling establishment

31

and plant densities than with the spring seed buoy method, and should be utilized if funding and facilities exist for long-term seed storage. Because restoration site selection is critical, refinement of SAV habitat criteria for restored populations of Z. marina is needed. More research on how habitat criteria differ for restored eelgrass in relation to established populations, and the inclusion of additional parameters, such as sediment characteristics and wave exposure, are necessary in order to enhance restoration site selection. The role of long-term trends and regional events or extremes in SAV habitat conditions must be considered in restoration projects. Monitoring frequency and scale should be considered when planning restoration projects as it is crucial to provide sufficient resolution in order to explain observed changes in Z. marina shoot density and long-term survival. The ecological function of restored Z. marina beds should be considered when defining restoration success.

32

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Churchill, A. C. 1992. Growth characteristics of Zostera marina seedlings under anaerobic conditions. Aquatic Botany. 43:379-392.

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Evans, A. S., K. L. Webb and P. A. Penhale. 1986. Photosynthetic temperature acclimation in two coexisting seagrasses, Zostera marina L. and Ruppia maritima L. Aquatic Botany 24:185-197. Fishman, J. R. and R. J. Orth. 1996. Effects of predation on Zostera marina L. seed abundance. Journal of Experimental Marine Biology and Ecology. 198:11-26.

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Granger, S., M. Traber, S.W. Nixon and R. Keyes. 2002. Part 1. Collection, processing and storage. Pages 1-20 in M. Schwartz, editor. A practical guide for the use of seeds in eelgrass (Zostera marina L.) restoration. Rhode Island Sea Grant, Narragansett R.I.

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Harwell, M.C., and R.J. Orth. 1999. Eelgrass (Zostera marina L.) seed protection for field experiments and implications for large scale restoration. Aquatic Botany 64:51-61.

Holmer, M. and E. J. Bondgaard. 2001. Photosynthetic and growth response of eelgrass to low oxygen and high sulfide concentrations during hypoxic events. Aquatic Botany 70:29-38.

Kemp W. M., W. R. Boynton, J. C. Stevenson, R. R. Twilley, and J. C. Means. 1983. The decline of submerged vascular plants in upper Chesapeake Bay: Summary of results concerning possible causes. Marine Technology Society Journal 17:78-89.

Kemp, W. M., P. Bergstrom, E. Koch, L. Murray, J. C. Stevenson, R. Bartleson, V. Carter, N. B. Rybicki, J. M. Landwehr, C. Gallegos, L. Karrh, M. Naylor, D. Wilcox, K. A. Moore and R. Batiuk. 2004. Habitat requirements for submerged aquatic vegetation in Chesapeake Bay: water quality, light regime, and physical-chemical factors. Estuaries 27:363-377. Landwehr, J. M., J. T. Reel, N. B. Rybicki, H. A. Ruhl and V. Carter. 1999. Chesapeake Bay habitat criteria scores and the distribution of submersed aquatic vegetation in the tidal Potomac River and Potomac estuary, 1983-1997. U.S. Geologic Survey Open-File Report 99-219.

Luckenbach, M. W. and R. J. Orth. 1999. Effects of a deposit-feeding invertebrate on the entrapment of Zostera marina L. seeds. Aquatic Botany 62:235-247.

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Marsh, J. A., W. C. Dennison and R. S. Alberte. 1986. Effects of temperature on photosynthesis and respiration in eelgrass (Zostera marina L.). Journal of Experimental Marine Ecology 101:257-267.

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Moore, K.A., R. J. Orth, and J. F. Nowak. 1993. Environmental regulation of seed germination in Zostera marina L. (eelgrass) in Chesapeake Bay: effects of light, oxygen and sediment burial. Aquatic Botany 45:79-91.

Moore, K. A. and J. C. Jarvis. 2008. Environmental factors affecting recent summertime eelgrass diebacks in the lower Chesapeake Bay: implication for long-term persistence. Journal of Coastal Research SI 55:135-147.

Nejrup, L. B. and M. F. Pedersen. 2008. Effects of salinity and water temperature on the ecological performance of Zostera marina. Aquatic Botany 88:239-246.

Orth, R. J. and K. A. Moore. 1983a. Chesapeake Bay: An unprecedented decline in submerged aquatic vegetation. Science 222:51-53.

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Orth, R.J. and K.A. Moore. 1983b. Seed germination and seedling growth of Zostera marina L. (eelgrass) in the Chesapeake Bay. Aquatic Botany 15:117-131.

Orth, R. J., and K. A. Moore. 1984. Distribution and abundance of submerged aquatic vegetation in Chesapeake Bay: A historical perspective. Estuaries 7:531-540.

Orth, R. J. and K.A. Moore. 1986. Seasonal and year-to-year variations in the growth of Zostera marina L. (eelgrass) in the Lower Chesapeake Bay. Aquatic Botany 24:335-341.

Orth, R. J., M. L. Luckenbach, and K. A. Moore. 1994. Seed dispersal in a marine macrophyte: implications for colonization and restoration. Ecology 75:1927-1939.

Orth, R. J., M. C. Harwell, and J. R. Fishman. 1999. A rapid and simple method for transplanting eelgrass using single, unanchored shoots. Aquatic Botany 64:77-85.

Orth, R. J., M. C. Harwell, E. M. Bailey, A. Bartholomew, J. T. Jawad, A. V. Lombana, K. A. Moore, J. M. Rhode, and H. E. Woods. 2000. A review of issues of seagrass seed dormancy and germination: implications for conservation and restoration. Marine Ecology Progress Series 200:277-288.

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Orth, R. J., J. R. Fishman, M. C. Harwell, and S. R. Marion. 2003. Seed density effects on germination and initial seedling establishment in eelgrass Zostera marina in the Chesapeake Bay Region. Marine Ecology Progress Series 250:71-79.

Orth, R. J., J. Bieri, J. R. Fishman, M. C. Harwell, S. R. Marion, K. A. Moore, J. F. Nowak, and J. van Montfrans. 2006a. A review of techniques using adult plants and seeds to transplant eelgrass (Zostera marina L.) in Chesapeake Bay and the Virginia Coastal Bays. Pages 1-17 in S. F. Treat and R. R. Lewis, editors. Conference Proceedings from Seagrass Restoration: Success, Failure, and the Costs of Both. March 11, 2003. Sarasota, FL.

Orth, R. J., M. L. Luckenbach, S. R. Marion, K. A. Moore and D. J. Wilcox. 2006b. Seagrass recovery in the Delmarva Coastal Bays, USA. Aquatic Botany 84:26-36.

Orth, R. J., and S. R. Marion. 2007. Innovative techniques for large-scale collection, processing and storage of eelgrass (Zostera marina) seeds. ERDC/TN SAV-07-2. U.S. Army Corps of Engineers, Vicksburg, MS. Available from: http://el.erdc.usace.army.mil/elpubs/pdf/sav07-2.pdf

Orth, R. J., D. J. Wilcox, L. S. Nagey, A. L. Owens, J. R. Whiting, and A. Serio. 2008a. 2007 Distribution of Submerged Aquatic Vegetation in the Chesapeake Bay and Coastal Bays. Final Report for award CB973013-01-0 from the U.S. Environmental Protection

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Agency, Chesapeake Bay Program, Annapolis, MD. Available from: http://www.vims.edu/bio/sav/sav07

Orth, R., S. Marion, S. Granger and M. Traber. 2008b. Restoring eelgrass (Zostera marina) from seed: a comparison of planting methods for large-scale projects. ERDC/TN SAV-08-1. U.S. Army Corps of Engineers, Vicksburg, Mississippi. Available from: http://el.erdc.usace.army.mil/elpubs/pdf/sav08-1.pdf

Parham, T., L. Karrh. 1998. Maryland Submerged Aquatic Vegetation Restoration Targeting System. Final Report to National Oceanic and Atmospheric Administration.

Paling, E.I., M. van Keulen, K. D. Wheeler, J. Philips, R. Dyhrberg, and D. A. Lord. 2001a. Mechanical seagrass transplantation in Western Australia. Ecological Engineering 16:331-339.

Paling, E. I., M. van Keulen, K. D. Wheeler, J. Philips, R. Dyhrberg, and D. A. Lord. 2001b. Improving mechanical seagrass transplantation. Ecological Engineering 18:107113.

Pedersen, O., T. Binzer and J. Borum. 2004. Sulphide intrusion in eelgrass (Zostera marina L.). Plant Cell and Environment 27:595-602.

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Pfitzenmeyer, H. T. and K. G. Drobeck. 1963. Benthic survey for populations of softshelled clams, Mya arenaria, in the lower Potomac River, Maryland. Chesapeake Science 4:67-74.

Pickerell, C. H., S. Schott, and S. Wyllie-Echerverria. 2005. Buoy-deployed seeding: Demonstration of a new eelgrass (Zostera marina L.) planting method. Ecological Engineering 25:127-136.

Pickerell, C., S. Schott, and S. Wyllie-Echeverria. 2006. Buoy-deployed seeding: A new low-cost technique for restoration of submerged aquatic vegetation from seed. ERDC/TN SAV-06-2. U.S. Army Corps of Engineers, Vicksburg, MS. Available from: http://el.erdc.usace.army.mil/elpubs/pdf/sav06-2.pdf

Plus, M., J-M. Deslous-Paoli and F. Dagault. 2003. Seagrass (Zostera marina L.) bed recolonisation after anoxia-induced full mortality. Aquatic Botany 77:121-134.

Pregnall, A. M., R. D. Smith, T. A. Kursar and R. S. Alberte. 1984. Metabolic adaptation of Zostera marina (eelgrass) to diurnal periods of root anoxia. Marine Biology 83: 141147.

Rasmussen, E. 1977. The wasting disease of eelgrass (Zostera marina) and its effects on environmental factors and fauna. Pages 1-51 in C. P. McRoy and C. Helfferich, editors. Seagrass ecosystems: a scientific perspective. Marcel Dekker, Inc., New York.

41

Shafer, D. J., and P. Bergstrom. 2008. Large-scale submerged aquatic vegetation restoration in Chesapeake Bay. Status Report 2003-2006. ERDC/EL TR-08-20. U.S. Army Corps of Engineers, Vicksburg, MS. Available from: http://el.erdc.usace.army.mil/elpubs/pdf/trel08-20.pdf

Smith, R. D., A. M. Pregnall and R. S. Alberte. 1988. Effects of anaerobiosis on root metabolism of Zostera marina (eelgrass)-implications for survival in reducing sediments. Marine Biology 98:131-141.

Traber, M. S., S. Granger and S. Nixon. 2003. Mechanical seeder provides alternative method for restoring eelgrass habitat (Rhode Island). Ecological Restoration 21:231-214.

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Zimmerman, R. C., R. D. Smith and R. S. Alberte. 1989. Thermal acclimation and wholeplant carbon balance in Zostera marina L. (eelgrass). Journal of Experimental Marine Ecology 130:93-109.

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Figure 1. Map of Patuxent River, MD with restoration locations: Parrans Hollow (PH), Jefferson Patterson Park (JPP), Hungerford Creek (HC), Myrtle Point (MP) and Solomons Island (SI).

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Figure 2. Map of Potomac River, MD with restoration locations: Piney Point (PP), St. George Island (SGI), Cherryfield Point (CP), Sage Point (SP) and Kitts Point (KP). Red stars denote sites that were seeded after seeding efforts were stopped on the Patuxent River during this project. 44

Figure 3. Zostera marina seed collection locations (2003-2008) and the Piney Point Aquaculture Facility, St. Marys County, Maryland where seed processing, sorting and storage took place.

45

Figure 4. Mechanical harvest machine used for collection of Zostera marina seeds (Pristine Marine, M J McCook & Associates, La Plata, MD).

46

Figure 5. Mechanical seed spraying apparatus (C& K Lord, Inc.) used for fall seed dispersal. Seeds were broadcast using the sprayer in 2004 and 2005. Subsequent broadcasts were accomplished using manual dispersal due to the large amount of time needed to utilize the seed sprayer.

47

Figure 6. A) Maryland Department of Natural Resources staff assembling spring seed bags, B) Spring seed bag with attached cinderblock, C) Spring seed bag deployment, and D) Spring seed bag floating freely after deployment.

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2005

2006

2007

2008

A
CP-04 SP-04 SGI-04 CP-05 SP-05 SGI-05 SGI-06 SGI-S-06 CP-06

Percent Survival

100 80 60 40 20 0
O ct Fe b Ju n O ct Fe b O ct Fe b Ju n O ct Fe b Ju n Ju n O ct

2005
100 80

2006

2007

B
HC-04

Percent Survival

60 40 20 0
ct Ja n ct ct Ja n Ap r Ja n Ap r Ap r Ju l Ju l O O O Ju l

PH-04 SI-04 HC-05 MP-05 PH-05 PH-06

Figure 7. Mean percent survival of adult test plantings. A) Potomac River: CP Cherryfield Point, SP Sage Point, SGI St. George Island, SGI-S St. George Island South (adjacent to 2006 seeding plots). B) Patuxent River: HC- Hungerford Creek, PH Parrans Hollow, SI Solomons Island, MP Myrtle Point. Year designations on the graph represent the monitoring year. Year designations in legend represent the year planted (fall).

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2.5
Fall Broadcast Spring Seed Bag

2 mean shoot density (shoots m-2)

1.5

0.5

0 Sp 05

Su 05

Fa 05

Sp 06

Su 06

Fa 06

Sp 07

Su 07

Fa 07

Sp 08

Su 08

Fa 08

Time

4.5
2004 2005 2006 2007

3.5 mean shoot density (shoots m-2)

2.5

1.5

0.5

0 Sp 05

Su 05

Fa 05

Sp 06

Su 06

Fa 06

Sp 07

Su 07

Fa 07

Sp 08

Su 08

Fa 08

50

2.5
Patuxent Potomac

2 mean shoot density (shoots m-2)

1.5

0.5

0 Sp 05

Su 05

Fa 05

Sp 06

Su 06

Fa 06

Sp 07

Su 07

Fa 07

Sp 08

Su 08

Fa 08

Figure 8. Comparison of mean Z. marina shoot densities observed during monitoring events throughout the project. A) Z. marina shoot densities by seed dispersal method (fall broadcast and spring seed bag) B) Z. marina shoot densities by seed dispersal year (20042007) C) Z. marina shoot densities by river (Patuxent and Potomac). The number of plots monitored increased each year (n = 16, 29, 33 and 36 for 2005, 2006, 2007 and 2008, respectively) as new seed dispersal plots were established.

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Table 1. Annual comparison of Zostera marina seed collection, processing, storage and dispersal methods.
2003 Collection Collection method No. of collection days Z. marina yield (L) Collection rate (L/day) Processing and Storage Volume of Z. marina seeds processed (L) Viable Z. marina seeds remaining after storage (no. and (% of total)) Dispersal Seeds dispersed through spring seed bag method (%) Seeds dispersed through fall broadcast method (%) Manual 8 22796 2849 2004 Mechanical 9 89918 9991 2005 Mechanical 9 204482 22720 Manual 8 1451 181 2006 Mechanical 4 2467 617 Total 10 3918 392 2007 Mechanical 7 54510 7787 2008 Mechanical 6 39179 6530

N/A 345000 (16)

71.9 1058400 (7)

109.8 2527000 (20)

32.5 349888 (87)

48.8 540867 (21)

70.3 961567 (60)

0 100

92 8

71 29

38 62

6 94

0 100

52

Table 2. Repeated measures ANOVA summary table for main effects and interactions with time on adult eelgrass transplants.

Variable Year Site River Year*Time Site*Time River*Time Year*Site*Time

Num df/Den df 2/33 6/33 1/7 4/64 11/64 8/124 18/64

F 24.42 18.19 5.45 0.77 8.20 30.02 18.80

p <0.0001 <0.0001 0.0522 0.5474 <0.0001 <0.0001 <0.0001

Table 3. Univariate ANOVA results for transplant survival at each monitoring period following a significant interaction with time.

Monitoring Period (months after transplant) 1 6 12 1 6 12

Variable Site Site Site River River River

df effect 5 5 1 1 1 1

MS effect df error 0.0486 16.0771 0.0106 16.6245 0.1167 17.1977 0.0122 0.2287 0.3201 45.3465 37.9782 43

MS error 0.02346 0.00387 0.0678 0.02698 0.08362 0.06761

F 3.74 11.44 0.30 0.50 2.59 4.73

p 0.0195 <0.0001 0.5934 0.4048 0.1156 0.0351

53

Table 4. Compilation of all eelgrass restoration efforts in the Patuxent River by restoration site (2003-2006) Restoration Site Parrans Hollow Year 2004 Broadcast Method Fall Seed Broadcast Spring Seed Bag Size (Acres) 0.25 5 1 Total Acres 6.25 0.1 0.07 2 3 1 3 Total Acres 9.17 2 0.25 Total Acres 2.25 0.5 2.5 Total Acres 3 0.25 5 Total Acres 5.25 Total Acres 25.92 54 Number of Seeds 37,500 605,000 245,000 Total Number of Seeds 887,500 87,500 56,000 534,000 201,000 150,000 300,000 Total Number of Seeds 1,328,500 534,000 37,500 Total Number of Seeds 571,500 133,500 300,000 Total Number of Seeds 433,500 37,500 605,000 Total Number of Seeds 642,500 Total Number of Seeds 3,863,500 Seeds/Acre 150,000 121,000 245,000

Jefferson Patterson Park

2006 2005 2004 2003

Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast

875,000 800,000 267,000 67,000 150,000 100,000

Hungerford Creek

2005 2004

Fall Seed Broadcast Fall Seed Broadcast

267,000 150,000

Myrtle Point

2005 2004

Fall Seed Broadcast Spring Seed Bag

267,000 120,000

Solomons Island

2004

Fall Seed Broadcast Spring Seed Bag

150,000 212,000

Grand totals of seeding on Patuxent River as of 2006

Table 5. Compilation of all eelgrass restoration efforts in the Potomac River by restoration site (2003-2008) Restoration Site Piney Point Year 2004 2003 Broadcast Method Fall Seed Broadcast Fall Seed Broadcast Size (Acres) 0.50 3.00 Total Acres 3.50 1.0 0.67 0.66 1.0 0.34 0.25 0.2 1.25 1.00 5.00 0.25 Total Acres 11.62 0.67 1.0 2.50 0.50 2.50 2.50 0.25 Total Acres 9.92 5.00 5.00 55 Number of Seeds 150,000 300,000 Total Number of Seeds 450,000 400,000 134,000 132,000 270,000 35,000 262,000 160,000 275,000 200,000 605,000 75,000 Total Number of Seeds 2,548,000 134,000 270,000 1,210,000 100,000 275,000 275,000 37,500 Total Number of Seeds 2,301,500 605,000 605,000 Seeds/Acre 300,000 100,000

St. George Island

2008

Fall Seed Broadcast

2007 2006 2005 2004

Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast Spring Seed Bag Spring Seed Bag Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast

400,000 200,000 200,000 270,000 105,000 1,050,000 800,000 220,000 200,000 121,000 300,000

Cherryfield Point

2008 2007 2005 2004

Fall Seed Broadcast Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast Spring Seed Bag Fall Seed Broadcast

200,000 270,000 484,000 200,000 110,000 110,000 150,000

Sage Point

2004

Spring Seed Bag

121,000 121,000

Total Acres 10.00 Kitts Point 2005 Fall Seed Broadcast Spring Seed Bag 0.50 2.50 2.50 2.50 Total Acres 8.00 Total Acres 43.04

Total Number of Seeds 1,210,000 100,000 605,000 1,210,000 1,210,000 Total Number of Seeds 3,125,000 Total Number of Seeds 9,634,500 200,000 242,000 484,000 484,000

Grand totals of seeding on Potomac River as of 2008

56

Table 6. Mean eelgrass seeds observed as seedlings (% SD) at each restoration site.

2004 Spring Seed Bag Fall Seed Broadcast 0 0.1 0 0 0 0 0.3+0.2 0.5 0.2

River

Site

Dispersal Year and Method 2005 2006 Fall Fall Spring Seed Spring Seed Seed Bag Broadcast Seed Bag Broadcast 0.3 2.8+0.3 0

2007 Fall Spring Seed Seed Bag Broadcast

Patuxent Hungerford Creek Jefferson Patterson Park Myrtle Point Parrans Hollow Solomons Island Potomac Cherryfield Point Kitt's Point Piney Point Sage Point St. George Island

0.1 0.03 0.08 0 0 0.2+0.3

0.2

0 0.03+0.06

0 0

0.2

2.6

1.1

0.2

0.9

3.7

1.7

Table 7. Summary of mean ( SD) eelgrass seedling establishment, first year survival and long-term survival for River, Dispersal method, and Dispersal year. First year survival is calculated as the percentage of initial seedlings observed as shoots after one year of monitoring. Long-term survival is calculated as the mean number of shoots observed as a percentage of initial seedlings established.

Variable River Patuxent Potomac Dispersal Method Spring Seed Bag Fall Seed Broadcast Dispersal Year 2004 2005 2006 2007

mean seedling establishment

mean 1st year survival

mean long-term survival

0.611.1 0.600.97

00 3760

00 338750

0.461.0 0.721.0

1549 3654

42108 467922

0.120.16 0.961.3 0.340.42 1.91.8

1.42.9 22.352.5 10994.8 44.459.9

143400 19.947.5 15191622 44.459.9

57

Table 8. Results of repeated measures ANOVAs testing the effects of seed dispersal year and method on eelgrass shoot density over three monitoring events (spring, summer, fall).

Variable Year Time Year * Time Method Time Method*Time

Num df/Den df 3/32 2/60 5/60 1/34 2/63 2/63

F 3.49 3.90 4.07 4.48 6.58 4.71

p 0.0268 0.0256 0.0030 0.0417 0.0025 0.0124

Table 9. Univariate ANOVA results at each monitoring period for seed dispersal year and method following a significant interaction with time.

Time (Monitoring Period)

Variable

df effect

MS effect

df error MS error

spring summer fall spring summer fall

Year Year Year Method Method Method

3 2 3 1 1 1

0.5161 18.6793 0.0598 0.2098 18.4669 0.0083

5.4683 7.2759 5.4557 26.479 16.538 32.660

0.1326 3.6765 0.0284 0.1661 4.1674 0.0318

3.03 10.36 1.75 1.21 4.19 0.26

0.1232 0.0074 0.2640 0.2808 0.0569 0.6117

58

Table 10. Mean percent of established eelgrass seedlings observed as shoots at each restoration site after the first year of monitoring.

2004 Fall Spring Seed Seed Bag Broadcast N/A 0 0 0 0 0 7.5

River

Site

Dispersal Year and Method 2005 2006 Fall Fall Spring Seed Spring Seed Seed Bag Broadcast Seed Bag Broadcast 0 0 N/A

2007 Fall Spring Seed Seed Bag Broadcast

Patuxent Hungerford Creek Jefferson Patterson Park Myrtle Point Parrans Hollow Potomac Cherryfield Point Kitts Point Sage Point St. George Island
N/A

N/A

N/A N/A N/A 0 15.8 N/A N/A 141 176 41.9 0 113

6.2

20.6

Initial seedling establishment was not observed, percent of seedlings observed can not be calculated

Table 11. Mean eelgrass shoots observed as a percentage of seedlings established in fall of 2008.

2004 Fall Spring Seed Seed Bag Broadcast N/A 0 0 0 77.3 0 0

River

Site

Dispersal Year and Method 2005 2006 Fall Fall Spring Seed Spring Seed Seed Bag Broadcast Seed Bag Broadcast 0 0 N/A

2007 Fall Spring Seed Seed Bag Broadcast

Patuxent Hungerford Creek Jefferson Patterson Park Myrtle Point Parrans Hollow Potomac Cherryfield Point Kitts Point Sage Point St. George Island
N/A

N/A

N/A N/A N/A 0 12.8 N/A N/A 127 372 2,666 0 113

1,273

20.6

Initial seedling establishment was not observed, percent of seedlings remaining can not be calculated

59

Table 12. Results of Kruskal-Wallis ANOVAs for temporal water quality measurements for each year of the project (April-October).

Year 2004

Variable Temperature Salinity Dissolved Oxygen Turbidity Chlorophyll Temperature Salinity Dissolved Oxygen Turbidity Chlorophyll Temperature Salinity Dissolved Oxygen Turbidity Chlorophyll Temperature Salinity Dissolved Oxygen Turbidity Chlorophyll

H statistic 6607 13777 5502 21200 4500 2445 3262 839 16916 7525 178 23984 5936 6982 4308 14.67 941.8 2650 2015 5574

DF 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2

p <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0007 <.0001 <.0001 <.0001 <.0001

2005

2006

2007

60

Table 13. Annual cost comparisons of two methods of dispersing Zostera marina seeds.

Total annual costs Spring seed bag 2004 2005 2006 2007 Fall seed broadcast 2004 2005 2006 2007 2008

No. of viable seeds dispersed

Cost per seed dispersed

Cost per Acre

$48,194 $30,464 $21,413 $2,850

2,155,000 2,255,000 108000 17500

$0.02 $0.01 $0.20 $0.16

$4,473 $2,702 $39,654 $32,571

$125,616 $153,294 $110,056 $142,718 $117,708

374,500 1,802,500 349,500 540,000 961,567

$0.34 $0.09 $0.31 $0.26 $0.12

$67,085 $17,009 $62,979 $52,859 $24,483

A seeding density of 200,000 seeds per acre was used to calculate annual costs per acre.

61

APPENDIX A Continuous Monitor Data

14

A
12 daily minimum dissolved oxygen (g/L) 10

2004 2005 2006 2007

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

14

B
12 Daily minimum dissolved oxygen (g/L) 10

8 2004 2005 6

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 1. Daily minimum dissolved oxygen levels at continuous monitor stations A) Pin Oak and B) Chesapeake Biological Lab on the Patuxent River throughout the project. 62

14

A
12 daily minimum dissolved oxygen (g/L) 10

8 2004 2005 6

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

14

B
12 daily minimum dissolved oxygen (g/L) 10

2004 2005 2006 2007

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

63

14

C
12 daily minimum dissolved oxygen (mg/L) 10

8 2006 2007 6

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 2. Daily minimum dissolved oxygen levels at continuous monitor stations A) Sage Point B) Piney Point and C) St. George Creek on the Potomac River throughout the project.

64

35

A
30 Daily maximum water temperature (degrees C) 25

20

15

2004 2005 2006 2007

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

35

B
30 Daily maximum water temperature (degrees C) 25

20 2004 2005 15

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 3. Daily maximum water temperatures at continuous monitor stations A) Pin Oak and B) Chesapeake Biological Lab on the Patuxent River throughout the project.

65

35

A
30 Daily maximum water temperature (degrees C) 25

20 2004 2005 15

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

35

30

Daily maximum water temperature (degrees C)

25

20

15

2004 2005 2006 2007

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

66

35

C
30 Daily maximum water temperature (degrees C) 25

20 2006 2007 15

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 4. Daily maximum water temperatures at continuous monitor stations A) Sage Point B) Piney Point and C) St. George Creek on the Potomac River throughout the project.

67

150

A
125

Daily maximum turbidity (NTU)

100

75

2004 2005 2006 2007

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

50

45

40

35 Daily maximum turbidity (NTU)

30

25

2004 2005

20

15

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 5. Daily maximum turbidity at continuous monitor stations A) Pin Oak and B) Chesapeake Biological Lab on the Patuxent River throughout the project.

68

150

125

Daily maximum turbidity (NTU)

100

75

2004 2005

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

150

B
125

Daily maximum turbidity (NTU)

100

75

2004 2005 2006 2007

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

69

100

C
90 80

70 Daily maximum turbidity (NTU)

60

50

2006 2007

40

30

20

10

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 6. Daily maximum turbidity at continuous monitor stations A) Sage Point B) Piney Point and C) St. George Creek on the Potomac River throughout the project.

70

250

A
225 200

Daily maximum chlorophyll (g/L)

175

150 2004 2005 2006 2007

125

100

75

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

175

150

125 Daily maximum chlorophyll (g/L)

100 2004 2005 75

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 7. Daily maximum chlorophyll at continuous monitor stations A) Pin Oak and B) Chesapeake Biological Lab on the Patuxent River throughout the project.

71

150

A
125

Daily maximum chlorophyll (g/L)

100

75

2004 2005

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

175

150

125 Daily maximum chlorophyll (g/L)

100

75

2004 2005 2006 2007

50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

72

125

C
100

Daily maximum chlorophyll (g/L)

75

2006 2007 50

25

0 4/1 4/15 4/29 5/13 5/27 6/10 6/24 7/8 7/22 8/5 8/19 9/2 9/16 9/30 10/14 10/28

Figure 8. Daily maximum chlorophyll at continuous monitor stations A) Sage Point B) Piney Point and C) St. George Creek on the Potomac River throughout the project.

73

APPENDIX B Cumulative Frequency Data (Derived from Continuous Monitor data)

Figure 1. Cumulative frequency distribution of A) turbidity, B) chlorophyll, C) dissolved oxygen and D) temperature recorded at the Pin Oak continuous monitor station between July 1 to August 31 from 2004 to 2007.

74

Figure 2. Cumulative frequency distribution of A) turbidity, B) chlorophyll, C) dissolved oxygen and D) temperature recorded at the Chesapeake Biological Laboratory continuous monitor station between July 1 to August 31 from 2004 to 2005.

75

Figure 3. Cumulative frequency distribution of A) turbidity, B) chlorophyll, C) dissolved oxygen and D) temperature recorded at the Piney Point continuous monitor station between July 1 to August 31 from 2004 to 2007.

76

Figure 4. Cumulative frequency distribution of A) turbidity, B) chlorophyll, C) dissolved oxygen and D) temperature recorded at the Sage Point continuous monitor station between July 1 to August 31 from 2004 to 2005.

77

Figure5. Cumulative frequency distribution of A) turbidity, B) chlorophyll, C) dissolved oxygen and D) temperature recorded at the St. George Creek continuous monitor station between July 1 to August 31 from 2006 to 2007.

78

APPENDIX C Dataflow Data

April

May

June

July

August

September

October

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5

Figure 1. Turbidity data (NTU) from DATAFLOW cruises from April to October 2003 on the Patuxent River.

12.5 +

79

March

April

May

June

July

August

September

October

November

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5

Figure 2. Turbidity data (NTU) from DATAFLOW cruises from March to November 2004 on the Patuxent River.

12.5 +

80

April

May

June

Turbidity (NTU)

July

August

September

0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5 12.5 +

Figure 3. Turbidity data (NTU) from DATAFLOW cruises from April to September 2005 on the Patuxent River.

81

Figure 4. Turbidity (NTU) data from DATAFLOW cruises from April to November 2006 on the Patuxent River.

82

June

July

August

September

October

Figure 5. Turbidity data (NTU) from DATAFLOW cruises from June to October 2004 on the Potomac River.

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5 12.5 +

83

April

May

June

July

August

September

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5

Figure 6. Turbidity data (NTU) from DATAFLOW cruises from April to September 2005 on the Potomac River.

12.5 +

84

April

May

June

July

August

September

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5

Figure 7. Turbidity data (NTU) from DATAFLOW cruises from April to September 2006 on the Potomac River.

12.5 +

85

April

May

June

July

August

September

Turbidity (NTU)
0 - 2.5 2.5 - 5 5 - 7.5 7.5 - 10 10 - 12.5

Figure 8. Turbidity data (NTU) from DATAFLOW cruises from April to September 2007 on the Potomac River.

12.5 +

86