A

Figure 6: Schematic of seagrass TubAssay (A) and RopeAssay (B) to detect responses of plants to water quality and light-attenuation with increasing depth. Plants are placed in each container and changes between initial and final parameters are recorded. The effect of water quality on plant survival and growth is evaluated between sites by comparing the magnitude of the changes observed.

TUB ASSAY
The TubAssay was designed to test the suitability of a site for seagrass restoration as it performs well as a measure of the relative energy of a site. Preliminary results from this assay indicate that hydrodynamic regime may be more important than light availability, in determining the success of seagrass establishment in a North Carolina estuary. Plans are to test this assay for the duration of the growth season of each species, Zostera in winter and spring, and Halodule in summer and fall in NC. TubAssays will be repeated in year 2 to determine inter-annual variability. We will use Zostera seedlings - the potentially most sensitive life-stage. Six seedlings (3x2 arrangement) will be planted in plastic tubs filled with sieved and dried beach sand and allowed to recover from transplanting stress, before being placed out at selected sites based on known water quality and the natural distribution of seagrass, based on historic and recent presence of seagrass beds (1992 and 2002 aerial photos). At a subset of appropriate locations additional tubs will be deployed approx. 0.5m deeper than the edge of the existing seagrass bed. Sites will be visited at biweekly intervals and the same suite of measurements as for the indoor light experiment will be taken (Table 1). The same experimental design will be used for Halodule, with the exception that mature plants with two shoots, one of which is an apical shoot, will be used instead of seedlings; Halodule reproduces asexually in NC so seedlings are not available for this species.

Tub Assay Results

ROPE ASSAY
The RopeAssay is a substrate independent test of water quality on seagrass growth, and can be implemented rapidly and with minimal resources at numerous sites to test the suitability of water quality for seagrass survival and growth. Our approach is to hydroponically incubate whole seagrass plants at increasing water depths (0.5, 1.0, 1.5m) for two weeks and determine growth, as well as physiological state before and after the incubation period (Table 1). Three to five plants (seedlings or mature apical+1shoot segments) are placed in 1.9L clear plastic storage containers. Containers are modified by drilling many small holes to allow free exchange of surrounding water during deployment. Containers are attached to a rope by cable-ties at the three depths, measured from a surface float. The rope assay is deployed in about 2m depths, and anchored by a heavy cinderblock (Fig. 6b). Again, the same suite of measurements as described above is taken on each plant at the beginning and end of the incubation period. Differences in water quality at a variety of sites can be determined by changes in growth, or diminished physiological state, measured by PEA and leaf reflectance spectra, after the two-week incubation compared to a control group. Assays will be repeated twice at each location to ensure similar results, based on known initial conditions of the plants used in the assay.
The next phase of testing for the RopeAssay will involve transplanting seagrass to locations that do not currently support seagrass, and comparing those water quality conditions to areas that do support seagrass communities. Test sites under consideration include the Neuse River estuary, North River, and eastern Pamlico Sound.

Table 1: frequency of data collection for each parameter in the lab and field experiments.

Methods/Techniques	1. Light Gradient	2. TubAssay	2. RopeAssay
# shoots	Weekly	biweekly	initial/final
# leaves	Weekly	biweekly	initial/final
Leaf length	Weekly	biweekly	initial/final
Leaf width	Biweekly	monthly	initial/final
Plastochrone Interval	Monthly	N/A	final
PEA & JIP-test	Weekly	biweekly	initial/final
Reflectance-Spectra	Biweekly	monthly	initial/final
Leaf sample	Biweekly	monthly	initial/final
C:N:P	Biweekly	monthly	initial/final
Chl a extraction	Biweekly	monthly	initial/final
Sugar reserves	monthly	monthly	initial/final

The transplant experiment was started in December to take advantage of the natural recruitment of Zostera seedlings. Zostera recruits (n=5-6) will be transplanted to plastic tubs (Rubbermaid 2951 or similar) with sediment (coarse to fine sand) from the donor area in Middle Marsh (Location Y). After a week recovery from planting stress, the tubs will be transplanted to sites upstream and downstream of where seagrasses are found. Two sites in each section will be set up. At each site three tubs will be placed at 1m depth and marked with a small fishing float attached to line tied off at the tub to allow easy retrieval during the monitoring stage. Tubs will be separated a distance of no less than 10 m (Fonseca 1996) to insure independence. In addition to the six sites established in this fashion, I will also place out additional tubs at the donor site. These will be placed at the deep edge of the seagrass bed, and also at a depth 0.5m below this edge. Survival of seedlings at these deeper depths is hypothesized to be light limited. Monitoring will initially occur weekly, and intensity may decline with time to monthly measurements.

Rope Assay Results
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