An analytical technique was developed to measure adenine nucleotide levels (ATP, ADP, AMP) and adenylate energy charge (EC) in Zostera marina (eelgrass), a submerged marine angiosperm. A tissue comparison and seasonal survey provide baseline information on natural adenylate variability. The methodology developed can be suitably adapted to other macrophyte species as well. Plants were frozen, lyophilized, scraped free of epiphytes, and homogenized. Adenylates were extracted with boiling 1 mM EDTA + 5% (w/v) PVPP (pH 7.6), and assayed by enzymic conversion of AMP and ADP to ATP, followed by quantitative analysis of ATP via the firefly bioluminescent reaction. ATP, ADP, total adenylates (AT), and EC were highest in leaf tissue (photophosphorylating source), while all adenylates were lowest in root plus rhizome. Monthly time series with aboveground tissue show ATP concentration highest in August and lowest in April, corresponding to periods of senescence (decreased ATP utilization) and growth (increased ATP utilization), respectively. Response of adenine nucleotides and EC in Z. marina to nutrient enrichment, light reduction, and herbicide (atrazine) exposure was evaluated as a monitor of metabolic state. Nutrient enrichment over 2 weeks increased epiphyte colonization, which in turn, appeared to negatively impact Z. marina adenylate content, net productivity, and growth. Z. marina ATP, AT, and EC were weakly and positively correlated with nutrients and light, but decreased over time. Short-term (6 hr) atrazine stress reduced ATP and AT at both 10 and 100 ppb, but EC remained constant. Net productivity decreased at 100, but not at 10 ppb atrazine over 6 hrs. Long-term (21 day) atrazine stress was evident from growth inhibition and 50% mortality near 100 ppb. EC was reduced at 0.1, 1.0, and 10 ppb atrazine, but ATP and EC increased with physiological adaptation to severe stress (100 ppb) after 21 days. Apparently, ATP and AT decrease over the short-term but rebound over the long-term with severe atrazine stress, increasing beyond control levels before plant death results. Supplementing adenine nucleotide and EC results with more conventional quantitative analyses would afford greater knowledge of physiological response to environmental variation.

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The goal of this study was to determine the significance of dissolved inorganic nitrogen (DIN) and dissolved organic nitrogen (DON) to harmful phytoplankton. Two regions that experience frequent and persistent harmful algal blooms (HABs) were examined, the Eastern Gulf of Mexico and York River, Virginia. Nitrogen uptake by harmful algae in these regions was examined using a combination of stable isotopic (15N) tracer techniques and nutrient bioassays. In the Eastern Gulf of Mexico, kinetic parameters for uptake of N substrates by K. brevis were determined and indicated the greatest preference for ammonium (NH4+), although all substrates tested were taken up. Investigation of N uptake by K. brevis in the light and dark revealed periodicity of diet uptake rates with the maximum and minimum rates measured early in the light and dark periods, respectively. The highest rates of uptake were observed for NH4+. Ratios of NH4+ regeneration:uptake were ∼1, indicating the importance of regeneration processes to blooms. Three strains of K. brevis exhibited significant differences in N uptake rates. The observed nutritional flexibility of K. brevis likely helps it flourish under a range of conditions spanning bloom initiation in oligotrophic offshore waters to bloom maintenance inshore. In the York River, N uptake was dominated by NH4+ with the highest uptake rates at all stations, for all size fractions and for all seasons, ranging from 34 to 80% of total absolute uptake. Rates of N uptake by A. monilatum are the first reported for this species in the York River, and demonstrate uptake of a diverse suite N substrates. During the A. monilatum bloom NH4+ regeneration rates equaled those of uptake, indicating the importance of regeneration to blooms. Additionally, three anthropogenic N sources were used to assess their role in exacerbation of a HAB during a 7 day bioassay. Urban parking lot run-off (+ Urban), soil from a construction site (+ Soil) and paper mill run-off (+ Industrial) were added to a natural bloom assemblage. Results indicated the anthropogenic sources had unique N compositions; DIN comprised ∼9%, 91% and 20% of + Urban, + Soil, and + Industrial, respectively. All N sources stimulated the growth of phytoplankton with the + Urban and + Soil treatments eliciting the greatest response, a doubling in Chl a and/or cell concentrations along with nutrient drawdown of both DIN and DON within two days. The results of this dissertation emphasize the importance of a flexible metabolism to the success of the HAB species investigated here. All harmful phytoplankton studied were able to utilize the variety of DIN and DON sources supplied. Additionally, a universal preference for NH4+ was observed in all studies despite the distinct regions examined and unique characteristics of each species.

The effects of chlorinated sea water on the blue crab, Callinectes sapidus Rathbun, were evaluated through measurements of acute toxicity, serum constituents (protein, total ninhydrin positive substances, chloride, osmotic, sodium, potassium, calcium, and magnesium concentrations), oxygen consumption and ventilation rates in whole crabs, and oxygen consumption of excised gills. Histological examination of gills was also performed. The 96-hr LC50 for female blue crabs exposed to chlorinated sea water at 21�C and 14('0)/00 was 0.86 mgCl(,2)/l CIO (chlorine induced oxidants). The value for males under similar conditions was 0.84 mgCl(,2)/l CIO. Mortality was uncommon below 0.61 mgCl(,2)/l, but reached 100% with as little as 1.15 mgCl(,2)/l. Exposure of adult females to 0.79 mgCl(,2)/l CIO for two hours in a flow-through respirometer caused decreases in oxygen consumption. These decreases were interpreted as short-term reactions to sensing the chlorinated water, since 96-hr exposures of crabs to 0.99 MgCl(,2)/l CIO caused no consistent changes in statistically measured oxygen consumption of whole crabs or excised gills relative to controls. In addition, except for short-term reactions, the same levels of CIO had no effects on crab ventilation rates. Histological examination of gills revealed no observable damage from exposure to CIO. CIO levels ranging from 0.36 to 1.04 mgCl(,2)/l had no consistent effects on any of the serum constituents, except for magnesium. Hyporegulation of this ion was disrupted (serum magnesium concentration increased relative to controls and approached external medium levels) during 96-hr exposures of crabs to CIO levels of 0.47 mgCl(,2)l and higher. The magnitude of the effect was directly related to dose. The effect of CIO on blue crab serum magnesium is the same as that found for other crabs exposed to CIO and chlorinated hydrocarbon pesticides. It is hypothesized that increased serum magnesium levels interfere with neural activity, causing death. Since magnesium regulation in the blue crab occurs at the bladder wall, the implication is that CIO acts on the crab at this internal site. This possibility compounds the already difficult problem of toxicant identification when using CIO.

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