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Quantifying the Magnitude of Nutrient Limitation on Phytoplankton in Kings Bay, Florida, USA


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QUANTIFYING THE MAGNITUDE OF NUTRIENT LIMITATION ON PHYTOPLANKTON IN KINGS BAY, FLORIDA, USA By DARLENE SAINDON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Darlene Saindon

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This document is dedicated to my family: Mike, Jeannie, Anna, and Travina, and to the past, present, and future generations of sta ff and graduate students of the Frazer Lab at the University of Florida, especially St eph, Sky, Kelly, Kristin, Vince, Emily, and Ray.

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ACKNOWLEDGEMENTS I thank my committee members for their support and guidance. I also thank the students and staff of the University of Floridas Department of Fisheries and Aquatic Science for use of their equipment and their assistance in running this experiment. Funding for this project was provided by the University of Floridas Institute of Food and Agricultural Science and the Swisher Water Resources Graduate Research Assistantship. iv

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TABLE OF CONTENTS page ACKNOWLEDGEMENTS ...............................................................................................iv LIST OF TABLES .............................................................................................................vi LIST OF FIGURES ..........................................................................................................vii ABSTRACT .....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 STUDY LOCATION....................................................................................................5 3 METHODS...................................................................................................................7 Water Collection...........................................................................................................7 Laboratory Assays of Nutrient Limitation....................................................................7 Chlorophyll Analysis....................................................................................................9 Nutrient Analysis........................................................................................................10 Calculation of Growth Rates and Maximum Biomass...............................................10 Statistical Analysis......................................................................................................10 Nutrient Limitation Assessment.................................................................................11 4 RESULTS...................................................................................................................13 Ambient Conditions....................................................................................................13 Experimental Assay....................................................................................................14 5 DISCUSSION.............................................................................................................23 Comparing Ambient and Experimental Results.........................................................23 Experimental Assay Implications...............................................................................23 LIST OF REFERENCES...................................................................................................28 BIOGRAPHICAL SKETCH.............................................................................................32 v

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LIST OF TABLES Table page 3-1 Experimental design with bioavailable N:P ratios at the start of the assays for water collected from sites 23 (a) and 81(b)..............................................................12 4-1 Summary of ambient water quality parameters for sites 23 and 81............................16 4-2 Summary of statistical results for the overall effects of nitrogen and phosphorus on phytoplankton growth rates and maximum biomass................................................17 4-3 Summary of statistical results for the effects of nitrogen and phosphorus on phytoplankton maximum biomass at fixed levels of the other nutrient...................18 vi

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LIST OF FIGURES Figure page 2-1 Map of Kings Bay..........................................................................................................6 4-1 Two dimensional growth rate response surface for site 23.........................................19 4-2 Two dimensional growth rate response surface for site 81.........................................20 4-3 Two dimensional maximum biomass response surface for site 23.............................21 4-4 Two dimensional maximum biomass response surface for site 81.............................22 vii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science QUANTIFYING THE MAGNITUDE OF NUTRIENT LIMITATION ON PHYTOPLANKTON IN KINGS BAY, FLORIDA, USA By Darlene Saindon December 2005 Chair: Tom Frazer Co-Chair: Craig Osenberg Major Department: Fisheries and Aquatic Sciences The recreational and economic viability of many aquatic systems is linked to water quality in general and water clarity in particular. In cases where water clarity has been impaired as a consequence of excessive phytoplankton production, a common management strategy is to reduce the load of the limiting nutrient. In this study, nutrient addition and dilution assays were used to quantify the magnitude of nutrient (nitrogen and phosphorus) limitation, and characterize the two-dimensional response surface of phytoplankton across a broad range of nutrient concentrations and N:P ratios. Site water from two sites in Kings Bay, Florida (USA) was filtered using a Millipore stirred cell concentrator, which allowed a fixed percentage of site water and nutrients to be removed while maintaining ambient abundances of plankton. DI water and stock solutions of nitrogen and phosphorus were added to return samples to their original volumes and create nutrient concentration treatments that ranged from ~ to 10 times the ambient concentration of phosphorus, and ~ to 15 times the ambient concentration of nitrogen. viii

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Growth rates and maximum biomass were estimated using in vivo fluorescence measures. The magnitude of limitation was quantified by estimating how much the response variable changed per unit of nutrient concentration. Phytoplankton growth rates were not limited by nitrogen or phosphorus at one site, but limited by both nitrogen and phosphorus at the other site. The overall estimated magnitude of limitation for this latter site was a 0.1011 change in per capita algal growth (d -1 ) for each mg L -1 of nitrogen, and a 1.1268 change in per capita algal growth (d -1 ) for each mg L -1 of phosphorus. Neither site showed significant nitrogen/phosphorus interaction effects on growth rate responses. However, there was a significant interactive effect of nitrogen and phosphorus on maximum biomass responses at both sites. A qualitative assessment of the response surfaces for maximum biomass suggests that this variable is affected by both the N:P ratio of nutrient treatments as well as the total amount of nutrients supplied. Since the magnitude of limitation of one nutrient is determined by the concentration of the other nutrient, an overall magnitude of limitation may either overestimate or underestimate the expected maximum biomass response for specific co-occurring changes in nutrient concentrations. Instead, magnitude of limitation should be determined on a case by case basis, taking into consideration specific nutrient remediation scenarios and creating a response surface with those co-occurring changes in nutrient concentrations in mind. The combination of quantitative analyses and characterization of two-dimensional response surfaces can be used by water resource managers as a tool to develop and implement nutrient reduction strategies with predictable outcomes. ix

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CHAPTER 1 INTRODUCTION Eutrophication of aquatic ecosystems, as a consequence of increased nutrient delivery, occurs worldwide. It can result in an increase in the frequency and intensity of algal blooms and fish kills, as well as a decrease in biodiversity with losses of key species such as rooted aquatic plants and corals (Grall and Chauvaud 2002, Baird et al. 2004, Hauxwell et al. 2004, Lapointe et al. 2004, Mller and Stadelmann 2004). The resulting decline in water quality and ecosystem function can have pronounced socioeconomic impacts that negatively affect a large number of recreational, agricultural, and industrial interests (Lund 1972, Carpenter et al. 1998, Smith et al. 1999, Tett et al. 2003). In response to the many issues related to cultural eutrophication, there has been a concerted effort on the part of scientists and water resource managers to improve water clarity and reverse the effects of eutrophication. Nutrient reduction strategies, however, have resulted in only limited success. In fact, many systems show no response to nutrient reduction. Carpenter et al. (1999), for example, showed that the effects of prior nutrient enrichment could be (1) reversible (recovery is immediate and proportional to the nutrient reduction), (2) hysteretic (recovery requires extreme reductions in nutrient input for a period of time), or (3) irreversible (recovery cannot be accomplished by reducing nutrient input alone). To determine why hysteretic and irreversible systems do not respond to nutrient remediation and reduction strategies, scientists and managers are now focused on better understanding the complex suite of physical, chemical, and biological interactions that affect nutrient uptake and assimilation. 1

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2 An understanding of the relationship between phytoplankton abundance and nutrient availability is vital to our ability to predict the effects of either nutrient increases or reductions. This relationship is especially important for systems where phytoplankton are one of the primary factors reducing water clarity. Of particular importance is the ability to determine whether or not phytoplankton growth is limited by a particular nutrient and identifying what that nutrient is. Nutrient limitation may be inferred from an analysis of nutrient availability and elemental ratios in phytoplankton or bulk suspended material in the water column. A more direct experimental evaluation can be made by adding nutrients to a water sample and evaluating if the growth, biomass, or other physiological parameter (e.g., respiration or carbon fixation rate) of phytoplankton differs significantly from a control treatment (Gerhart and Likens 1975, Beardall et al. 2001). Results from these types of studies can yield important insights to the current limiting status of a system, but may be of limited utility to managers that need to make quantitative predictions of a systems behavior to specific management actions. There are three primary limitations of this simple experimental approach. First, it focuses on two nutrient concentrations (added and control), which may or may not be relevant to the situations of interest to the managers. Second, many studies are not able to capture any complex interactions that may be occurring among nutrients. Some studies focus on the manipulation of a single nutrient, whereas others have limited nutrient treatment combinations (2x2 factorial with added and control treatments for each nutrient). In either case, interactions among nutrients can not be characterized. By manipulating >1 nutrient, all at a range of concentrations, multi-dimensional response surfaces can be obtained and characterized to reveal potentially complex nutrient

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3 interactions. Finally, the standard experimental approach focuses on the detection of statistical differences between treatments, which may not reveal biologically important effects which may or may not be statistically significant (e.g., Stewart-Oaten 1996). Instead, quantitative estimates of the magnitude of limitation (e.g., Downing et al. 1999, and Osenberg et al. 2002) may be more useful because these estimates reveal how much systems might respond to changes in nutrients. As a result, scientists and managers can directly test responses of phytoplankton to the range of nutrient concentrations they are currently exposed to or may be exposed to in the future. This information may provide managers with the detailed information necessary to implement policies that have an increased probability of success. This study introduces a method combining nutrient addition and dilution assays to create a two-dimensional response surface, and thus quantify the magnitude of nitrogen and phosphorus limitation. Both phytoplankton growth rates and maximum achieved biomass were measured to better understand how phytoplankton might respond to changes in nitrogen and phosphorus concentrations. Conceptually, there are four possible qualitative outcomes of this experiment based on the two response variables: (1) nutrients are never limiting, i.e. initial growth rates and maximum achieved biomass are similar across all nutrient treatments, (2) nutrients are not limiting at first, but become limiting as nutrients are depleted, i.e. initial growth rates are similar across treatments, but maximum achieved biomass increases with increasing nutrient concentration, (3) the nutrient is limiting at first, but some other factor consistent across treatments becomes limiting when the culture reaches a particular biomass, i.e. initial growth rates increase with increasing nutrient concentration, but maximum achieved biomass are similar across all

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4 nutrient treatments, and (4) the nutrient is always limiting; i.e., both initial growth rates and maximum achieved biomass increase with increasing nutrient concentration. The potential effects of nutrient manipulation on phytoplankton growth and biomass have seldom been quantified. Thus, the methodology and results reported herein will be of interest to a broad group of scientists and water resource managers.

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CHAPTER 2 STUDY LOCATION Kings Bay (Figure 2-1) is a tidally influenced system (ca. 1.75 km 2 ; see Haller et al. 1983) that is connected to the Gulf of Mexico by Crystal River on the west coast of Florida, about 96-km north of Tampa (Frazer et al. 2001). Greater than 30 springs in Kings Bay have a combined average total discharge of ~27 m 3 s -1 accounting for approximately ninety-nine percent of the freshwater entering the system (Yobbi and Knochenmus 1989, Hammett et al. 1996). Over 100,000 boaters, anglers, and sport divers visit these springs each year (USFWS 2005). Kings Bay is within the confines of the Crystal River National Wildlife Refuge and provides valuable habitat for a variety of native and endangered species. Since 1999, greater than 250 West Indian manatees (Trichechus manatus) take refuge annually in the relatively warm spring waters during winter months (J. Kleen, USFWS, pers. comm.). This system is a recreationally, economically, and ecologically important area that relies on maintaining water clarity and functional habitats to continue being utilized in this manner. As part of the Surface Water Improvement and Management (SWIM) program, one of the primary management goals for Kings Bay is to improve water clarity and wildlife habitat (SWFWMD 2003). Hoyer et al. (1997) found that the water clarity in Kings Bay is primarily determined by algal suspended particles, and moderately affected by non-volatile and detrital suspended particles. In an effort to reduce eutrophication, point sources of nutrients (e.g. effluent from the City of Crystal River Sewage Treatment Plant) were removed in 1992. As a result, average total phosphorus was reduced from 105 ug 5

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6 L -1 to 27 ug L -1 and average total nitrogen was reduced from 620 ug L -1 to 220 ug L -1 in Cedar Cove located in the northern region of Kings Bay (Terrell and Canfield 1996). Water clarity, however, has not improved (Bishop and Canfield 1995, Hoyer et al. 1997, SWFWMD 2003). Figure 2-1 Map of Kings Bay with sampling sites. There are > 30 springs located throughout Kings Bay with the main spring for Kings Bay located in the southern region, southeast of Site 81. Prior to 1992, the Crystal River Sewage Treatment Plant discharged effluent into Cedar Cove in the northeast region of Kings Bay. The dark grey/ black areas surrounding the bay correspond to developed coastline, whereas the light grey areas correspond to marshland.

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CHAPTER 3 METHODS Water Collection Two sites within Kings Bay were selected for water collection and subsequent nutrient manipulation. These two sites, numbers 23 and 81 (see Figure 2-1), correspond geographically to those established by Frazer and Hale (2001) as part of a longer-term vegetative monitoring program (see Notestein et al. 2005). On October 11, 2004, surface water samples (~ 0.5 m depth) from both sites were collected and analyzed within 24h for nitrate + nitrite (NO 3 + NO 2 ) and soluble reactive phosphorus (SRP) to determine ambient concentrations. These values were then used to estimate the amount of stock solutions needed to create designated treatments (see Table 3-1). The next day, water was again collected at each site (20-L samples). Water samples were transported immediately, in covered carboys, to the laboratory and allowed to set in the dark overnight in an incubation room before initiation of the experiment the following morning. Laboratory Assays of Nutrient Limitation Phytoplankton growth rate (d -1 ) and maximum algal biomass, as indicated by in vivo fluorescence (IVF) measures of chlorophyll-a, were determined over a gradient of nitrogen and phosphorus concentrations. The experiment was set up as a fully crossed factorial design, with 5 manipulated nitrogen and 5 manipulated phosphorus treatments. Each treatment was replicated three times. Target phosphorus treatments corresponded approximately to ambient SRP (X), 50% dilution (0.5 X), 25% dilution (0.75 X), 25% addition (1.25 X), and 50% addition (1.5 X). Nitrogen treatments were chosen to 7

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8 correspond to ambient nitrate + nitrite (X), 50% dilution (0.5 X), and additions that were double (2 X), four times (4 X), and eight times (8 X) ambient, reflecting a particular interest in and concern for increased nitrate in this and other spring-fed systems in Florida (SWFWMD 2003). An additional control (run in triplicate) that lacked a nutrient manipulation was used to determine ambient conditions and verify that the responses of phytoplankton to experimental nutrient manipulation were realistic. Because the target nutrient treatment levels were chosen based on water analyses run the previous day, the ambient nutrient levels (X) were not necessarily identical to the nutrient levels in the ambient control treatment. To establish the nutrient gradients, water collected from the two sites was filtered using a Millipore stirred cell concentrator (model 8400), with Biomax-30 filters (NMWL: 30,000). This allowed a pre-determined percentage of site water and nutrients to be removed while maintaining ambient abundances of plankton. For all replicates within a treatment, except the ambient control, a 400-ml aliquot of site water was filtered to 50% of its original volume to control for filtration effects. The unfiltered portion, with ambient concentration of plankton, was then placed in a 500-ml Erlenmeyer flask. DI water and stock solutions of known nitrogen (KNO 3 ) and phosphorus (K 2 HPO 4 ) concentration were added to return samples to the original volume of 400 ml and establish the nutrient concentration treatments (see Table 3-1). For the ambient control, site water was filtered to 50% of the original volume to control for filtration effects, and the filtrate, rather than DI water, added back to the sample to maintain both ambient nutrient and plankton concentrations. This control was used to determine ambient growth rate and biomass, and provided a comparison to determine if there was an effect of

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9 removing micronutrients from the experimental assays. Within 5 min of the nutrients being added, 100 ml of sample water from each flask was withdrawn and later analyzed for initial chlorophyll-a and nutrient concentrations. Nutrient measurements include total nitrogen (TN), nitrate + nitrite (NO 3 +NO 2 ), ammonium (NH 4 + ), total phosphorus (TP), and soluble reactive phosphorus (SRP). The nitrogen and phosphorus treatments reported herein are the averaged initial nitrate + nitrite and SRP concentrations measured from the assays. All Erlenmeyer flasks were subsequently incubated in temperature-controlled water baths with bottom illumination (~120 Em -2 s -1 ). Incubation temperatures were maintained at ambient temperatures (24C) recorded on the water collection date with a photoperiod of 11/13 dark/light hours. Algal biomass was estimated using IVF measures at the time of nutrient addition (time 0), and at 12 h intervals thereafter until IVF values in all experimental flasks had peaked. IVF was measured using a Turner Designs Model 10 fluorometer with a 1-cm path length. At the end of the incubation period, water remaining in the flasks was used for final IVF, chlorophyll-a, and nutrient measurements. Chlorophyll Analysis Initial and final chlorophyll-a measurements were obtained by filtering a minimum of 50 ml of sample water through a 47-mm Whatman GF/F filter. Filters were stored over desiccant and frozen prior to analysis. Chlorophyll-a was extracted in a 90% heated ethanol solution, and chlorophyll-a concentrations were determined spectrophotometrically using a Digilab Hitachi U-2810 spectrophotometer with a 4-cm path length (APHA 1998).

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10 Nutrient Analysis Total nitrogen and total phosphorus concentrations were determined from whole water samples digested with potassium persulfate. Ammonium, nitrate + nitrite, and SRP were determined from water samples filtered through a 47-mm Whatman GF/F filter. Total nitrogen, ammonium, and nitrate + nitrite analyses were determined spectrophotometrically on a Bran-Luebbe autoanalyzer with a cadmium column reduction method. Total phosphorus and SRP were determined spectrophotometrically on a Digilab Hitachi U-2810 spectrophotometer with a 4-cm path length (APHA 1998). Calculation of Growth Rates and Maximum Biomass Phytoplankton growth rates (d -1 ) were estimated as ln(IVF t /IVF 0 )/t, where IVF 0 was the initial fluorescence and IVF t was the fluorescence after t days, where t was determined visually as the time period during which ln(IVF) was an approximately linear function of time. Maximum biomass was determined as the peak fluorescence measured just before IVF began to decrease as the algal assemblage crashed. This peak sometimes coincided with the end of exponential growth, but usually lagged by 0.5-1.5 days. Statistical Analysis Because nitrogen and phosphorus treatments were not set at the same concentrations for the two sites, an analysis of co-variance (ANCOVA, with [N] and [P] as the covariates) was used to determine whether the two sites showed similar responses to nutrient treatments. An analysis of variance (ANOVA) was then run for each site individually to determine how nutrient treatments affected phytoplankton growth rates and maximum biomass. For all analyses, the simplest model was used by removing higher order treatment effects that were not significant; nutrient effects were determined to be significant at = 0.05 (SAS Institute 2004).

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11 Nutrient Limitation Assessment The magnitude of nutrient limitation is reflected as the change of the response variable (i.e. growth rate or maximum biomass) for a given unit change of nitrogen or phosphorus concentration. Graphically, the magnitude of limitation is the slope of the relationship between growth rate (or maximum biomass) and nutrient concentration. If there is no interaction between nitrogen and phosphorus, then the magnitude of limitation for one nutrient can be expressed independently of the concentration of the other nutrient. Where there was no nitrogen/phosphorus interactions, estimates of the treatment effects determined by regression analysis were used as the overall estimate of magnitude of limitation. If there is an interaction between N and P, then the magnitude of limitation for one nutrient is influenced by the concentration of the other nutrient. Where there were nitrogen/phosphorus interactions, estimates of treatment effects were also determined using regression analysis for nutrients at each concentration level of the other nutrient.

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12 Table 3-1 Bioavailable N:P ratios at the start of the assays for water collected from sites 23 (a) and 81 (b). N:P ratios < 7 (shaded) suggest nitrogen limited conditions; N:P ratios > 7 (unshaded) suggest phosphorus limited conditions. Nitrogen and phosphorus treatments reported are the averaged beginning nitrate + nitrite and SRP concentrations for each target treatment (n=15). Standard deviations are given in parentheses and the experimental treatments closest to ambient nutrient concentrations for each site are indicated with an asterisk. (a) Site 23 Nitrogen (ug/L) 25 (7.0) 65 (2.3) 167 (11.1) 385 (9.6) 794 (20.6) 7 (1.6) 3.57 9.29 23.86 55.00 113.43 12 (1.1) 2.08 *5.42 13.92 32.08 66.17 18 (1.0) 1.39 3.61 9.28 21.39 44.11 25 (1.0) 1.00 2.60 6.68 15.40 31.76 Phosphorus (ug/L) 32 (1.1) 0.78 2.03 5.22 12.03 24.81 (b) Site 81 Nitrogen (ug/L) 7 (3.8) 83 (3.6) 191 (8.1) 393 (23.3) 802 (42.4) 4 (1.4) 1.75 20.75 47.75 98.25 200.50 9 (1.2) 0.78 *9.22 21.22 43.67 89.11 16 (1.1) 0.44 5.19 11.94 24.56 50.13 23 (1.1) 0.30 3.61 8.30 17.09 34.87 Phosphorus (ug/L) 30 (1.1) 0.23 2.77 6.37 13.10 26.73

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CHAPTER 4 RESULTS Ambient Conditions Sites 23 and 81 had similar temperature, salinity, dissolved oxygen, and pH, but different nutrient concentrations (see Table 4-1). Ambient nitrate + nitrite and SRP concentrations at site 23 (83 ug L -1 and 11 ug L -1 respectively) were approximately twice those at site 81 (39 ug L -1 and 6 ug L -1 respectively). The bioavailable N:P ratio (ug:ug; nitrate + nitrite:SRP) at site 23 was 7.6, while at site 81 the N:P ratio was 6.5. Both sites 23 and 81 were near the expected 7:1 N:P weight ratio for the chemical composition of phytoplankton (Redfield 1934, Duarte 1992). This 7:1 N:P ratio is generally considered the threshold where phytoplankton switch from nitrogen limitation (N:P < 7) to phosphorus limitation (N:P > 7). Qualitative assessment of the plankton community suggests that both sites were comprised predominately of small centric diatoms and cryptophytes. There was, however, a lower concentration of chlorophyll-a at site 23 (10.92 ug L -1 ) than at site 81 (14.59 ug L -1 ). By using the model created by the experimental treatments, the ambient control responses can be compared to the experimental treatment responses to determine if the methodology may have affected the results. The maximum biomass achieved in the ambient control assays were 0.8023 for site 23 and 0.6117 for site 81, as indicated by IVF measures. By using the regression model created from the experimental assays (see Table 4-2), the maximum biomass (SE) for site 23 was expected to be 0.7042 0.1118, and site 81 was expected to be 0.7121 0.1187. Since the actual maximum biomass 13

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14 measured in the ambient control assays fall within these ranges, the methodology did not appear to bias the maximum biomass results. On the other hand, ambient growth rates for phytoplankton at sites 23 and 81 were 0.3494 and 0.4836 d -1 respectively. Again, by using the regression model created from the experimental assays (see Table 4-2), the growth rate (SE) for site 23 was expected to be 0.3019 0.0300, and site 81 was expected to be 0.4318 0.0400. Growth rates measured in the ambient control assays were greater than the expected, suggesting that the methodology employed may have slightly biased the growth rate results. Experimental Assay Mean algal growth rates in the assays ranged from 0.2493 to 0.3406 d -1 for site 23, and 0.3826 to 0.5349 d -1 for site 81 (Figures 4-1 and 4-2, respectively). The mean maximum biomass achieved across treatments, as indicated by IVF measures, ranged from 0.5667 to 1.0411 for site 23, and 0.6167 to 1.0179 for site 81 (Figures 4-3 and 4-4, respectively). There was a significant site effect on growth rate (F 2, 142 = 939.47, P < 0.0001) and maximum biomass (F 2, 138 = 161.89, P < 0.0001). In addition, there was a significant site and nitrogen treatment interaction effect (F 1, 142 = 5.18, P = 0.0243) on growth rate, as well as significant nitrogen x phosphorus (F 1, 138 = 13.99, P = 0.0003) and nitrogen 2 x phosphorus (F 1, 138 = 12.53, P = 0.0005) effects on maximum biomass. As a consequence of the significant site effects, phytoplankton responses to nutrient treatments were assessed for each site individually. Nitrogen and phosphorus had significant effects on phytoplankton growth rates only at site 81 (Table 4-2; Figures 4-1 and 4-2). The overall magnitude of nitrogen limitation (SE) at this site was estimated as 0.1011 0.0160 L mg -1 d -1 (i.e. a 10% increase in growth for each 1 mg L -1 increase in nitrogen concentration). Magnitude of

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15 limitation for phosphorus (SE), at site 81, was estimated as 1.1268 0.4866 L mg -1 d -1 For site 23, the estimated magnitude of limitation (SE) by both nitrogen (-0.0112 0.0120) and phosphorus (-0.1899 0.3792) included zero; hence the absence of a significant effect of the nitrogen or phosphorus gradient. With regard to the accumulation of phytoplankton biomass (Figures 4-3 and 4-4), there was a significant interaction between nitrogen and phosphorus on the maximum biomass response at both sites (see Table 4-2). Due to this interaction, estimates of the treatment effect were determined for each nutrient at fixed levels of the other nutrient (see Table 4-3). Treatment effects of phosphorus at fixed levels of nitrogen ranged from -0.9333 to 9.9987 (IVF) L mg -1 for site 23, and -2.3460 to 10.2664 (IVF) L mg -1 for site 81. Treatment effects of nitrogen at fixed levels of phosphorus ranged from 0.4786 to 2.0827 (IVF) L mg -1 for site 23, and 0.2339 to 1.6421 (IVF) L mg -1 for site 81. These results are consistent with the potential role of both nitrogen and phosphorus on phytoplankton responses as indicated with the growth rate results above. Qualitative assessment of the response surfaces for maximum biomass suggests that this variable is affected by both the N:P ratio of nutrient treatments as well as the total amount of nutrients supplied. Maximum biomass appeared to be maintained at low concentrations when the N:P weight ratios were either very low (N:P < 3) or very high (N:P > 27). At the lowest concentrations of each nutrient (i.e. 7 ug L -1 phosphorus and 25 ug L -1 nitrogen for site 23; 4 ug L -1 phosphorus and 7 ug L -1 nitrogen for site 81), maximum biomass does not significantly change regardless of the concentration of the other nutrient. When the N:P weight ratios were between 3 and 27, maximum biomass appeared to increase with increasing nutrient availability.

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16 Table 4-1 Summary of ambient water quality parameters for sites 23 and 81. Temperature, salinity, dissolved oxygen, and pH were measured in the field at the time of collection. All other parameters were taken from a subsample of the ambient control treatments. Site 23 Site 81 Temperature (C) 24.18 24.15 Salinity (ppt) 0.81 0.69 Dissolved Oxygen (mg L -1 ) 6.3 6.99 pH 8.19 8.18 Total Nitrogen (ug L -1 ) 183 147 Nitrate + Nitrite (ug L -1 ) 83 39 Ammonia (ug L -1 ) 7 13 Total Phosphorus (ug L -1 ) 27 27 SRP (ug L -1 ) 11 6 Chlorophyll-a (ug L -1 ) 10.92 14.59 Predominant Species Composition centric diatoms, cryptophytes centric diatoms, cryptophytes Maximum Biomass (IVF) 0.8023 0.6117 Growth Rate (d -1 ) 0.3494 0.4836 Doubling rate (d) 2.0 1.4

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17 Table 4-2 Factorial ANOVA results for sites 23 and 81 including estimates and standard error (SE) of the treatment effects. Treatment effects are considered significant at 0.05 (shaded). The simplest model was used for each analysis by removing non-significant higher order treatment interactions. The model for growth includes nitrogen (N) and phosphorus (P) treatment effects. The model for maximum biomass also includes an interaction of nitrogen and phosphorus (N*P), a quadratic term for nitrogen (N*N), and an interaction of that quadratic term with phosphorus (N*N*P). GROWTH Treatment Effect Estimate of Effect SE F 1, 72 -value P-value N -0.0112 0.0120 0.88 0.3518 Site 23 P -0.1899 0.3792 0.25 0.6180 Treatment Effect Estimate of Effect SE F 1, 72 -value P-value N 0.1011 0.0160 40.05 < 0.0001 Site 81 P 1.1268 0.4866 5.36 0.0234 MAXIMUM BIOMASS Treatment Effect Estimate of Effect SE F 1, 69 -value P-value N 0.2088 0.4470 0.22 0.6419 N*N -0.0898 0.5256 0.03 0.8648 P -3.8406 2.7967 1.89 0.1741 N*P 56.7800 21.4759 6.99 0.0101 Site 23 N*N*P -64.8845 25.2536 6.60 0.0124 Treatment Effect Estimate of Effect SE F 1, 69 -value P-value N 0.1025 0.3821 0.07 0.7894 N*N -0.1015 0.4461 0.05 0.8206 P -2.3749 2.7789 0.73 0.3957 N*P 54.0362 20.2441 7.12 0.0095 Site 81 N*N*P -58.0074 23.6320 6.03 0.0166

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18 Table 4-3 ANOVA regression analysis results for sites 23 and 81 including estimates and standard error (SE) of the treatment effects of one nutrient at fixed levels of the other nutrient. Treatment effects are considered significant at 0.05 (shaded). Treatment effects include nitrogen (N) and the quadratic term for nitrogen (N*N) at fixed phosphorus levels (ug L -1 ); and phosphorus (P) treatment effects at fixed nitrogen levels (ug L -1 ). SITE 23 Phosphorus Treatment Effect Estimate of Effect SE F 1,13 -value P-value N 0.4786 0.3469 1.9 0.1928 7 N*N -0.3931 0.4079 0.93 0.3542 N 0.954 0.3195 8.92 0.0114 12 N*N -0.8705 0.3757 5.37 0.0390 N 1.4974 0.4520 10.97 0.0062 18 N*N -1.701 0.5315 10.24 0.0076 N 1.3684 0.5300 6.67 0.0240 25 N*N -1.3589 0.6232 4.75 0.0499 N 2.0827 0.5059 16.95 0.0014 32 N*N -2.2249 0.5949 13.99 0.0028 Nitrogen Treatment Effect Estimate of Effect SE F 1,13 -value P-value 25 P 1.9124 1.3931 1.88 0.193 65 P -4.9333 1.8949 6.78 0.0219 167 P 2.6687 3.3061 0.65 0.4341 385 P 9.9987 5.2922 3.57 0.0814 794 P 0.0392 2.1218 0.00 0.9856 SITE 81 Phosphorus Treatment Effect Estimate of Effect SE F 1,13 -value P-value N 0.2518 0.2260 1.24 0.287 4 N*N -0.2660 0.2638 1.02 0.3332 N 0.2339 0.3223 0.53 0.482 9 N*N -0.0613 0.3762 0.03 0.8733 N 1.7768 0.3537 25.23 0.0003 16 N*N -2.1123 0.4129 26.17 0.0003 N 1.0387 0.4317 5.79 0.0331 23 N*N -1.2083 0.5040 5.75 0.0337 N 1.6421 0.5455 9.06 0.0109 30 N*N -1.6164 0.6368 6.44 0.026 Nitrogen Treatment Effect Estimate of Effect SE F 1,13 -value P-value 7 P -2.346 1.2318 3.63 0.0792 83 P -0.2692 3 0.01 0.9227 191 P 10.2664 2.9691 11.96 0.0042 393 P 7.428 4.5433 2.67 0.126 802 P 4.0152 3.8804 1.07 0.3197

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19 712182532 2565167385794 00.10.20.30.40.50.6Growth Rate (d-1)Phosphorus (ug L-1)Nitrogen (ug L-1) Figure 4-1 Growth rates for site 23 at given phosphorus and nitrogen treatments (SE 0.0300). There were no significant nitrogen or phosphorus effects on phytoplankton growth rates (see Table 3-2). Colors represent growth rates within the indicated interval and are not intended to imply statistically significant differences.

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20 49162330 783191393802 00.10.20.30.40.50.6Growth Rate(d-1)Phosphorus(ug L-1)Nitrogen(ug L-1) Figure 4-2 Growth rates for site 81 at given phosphorus and nitrogen treatments (SE 0.0400). There was a significant nitrogen (F 1, 72 = 40.05, P < 0.0001), and phosphorus (F 1, 72 = 5.36, P = 0.0234) effect on phytoplankton growth rates (Table 3-2). Magnitude of limitation was estimated as a 0.1011 change in growth rate (d -1 ) for each mg L -1 change in nitrogen, and an estimated 1.1268 change in growth rate (d -1 ) for each mg L -1 change in phosphorus. Colors represent growth rates within the indicated interval and are not intended to imply statistically significant differences.

PAGE 30

21 712182532 2565167385794 00.20.40.60.811.2MaximumBiomass(IVF)Phosphorus(ug L-1)Nitrogen(ug L-1) Figure 4-3 Maximum biomass, as indicated by in vivo fluorescence (IVF), for site 23 at given phosphorus and nitrogen treatments (SE 0.1118). There was a significant nitrogen x phosphorus interaction effect (F 1, 69 = 6.99, P = 0.0101) as well as a significant nitrogen 2 x phosphorus interaction effect (F 1, 69 = 6.60, P = 0.0124; Table 3-2). Colors represent maximum biomass within the indicated interval and are not intended to imply statistically significant differences.

PAGE 31

22 49162330 783191393802 00.20.40.60.811.2MaximumBiomass(IVF)Phosphorus(ug L-1)Nitrogen(ug L-1) Figure 4-4 Maximum biomass, as indicated by in vivo fluorescence for site 81 at given phosphorus and nitrogen treatments (SE 0.1187). There was a significant nitrogen x phosphorus interaction effect (F 1, 69 = 7.12, P = 0.0095) as well as a significant nitrogen 2 x phosphorus interaction effect (F 1, 69 = 6.03, P = 0.0166; Table 3-2). Colors represent maximum biomass within the indicated interval and are not intended to imply statistically significant differences.

PAGE 32

CHAPTER 5 DISCUSSION Comparing Ambient and Experimental Results Maximum biomass did not appear to be affected by the methodology used in this study, the actual maximum biomass values measured in the ambient control were within the ranges predicted by the models created from the experimental assays. There was, however, a discrepancy between the ambient control and the expected values from the experimental assay models for growth rate responses; the models underestimated the actual responses of phytoplankton growth rates for both sites. This discrepancy may be due to the removal of micronutrients during the filtration process. Further experimentation would have to be done to evaluate this hypothesis and determine what other nutrients, if any, limit phytoplankton growth rates in Kings Bay. Experimental Assay Implications The results of this study provide important insights into how phytoplankton growth rates and biomass are affected by nitrogen and phosphorus in Kings Bay. Phytoplankton growth rates were not necessarily directly correlated with maximum biomass, i.e. growth rate did not determine the maximum achieved biomass, just how long it took to reach that biomass. The phytoplankton growth rate responses were spatially heterogeneous. Growth rates were either affected by both nitrogen and phosphorus (site 81) or were not affected by nitrogen or phosphorus (site 23). In addition, there was no significant nitrogen/phosphorus interaction, indicating that the N:P ratio did not have a strong 23

PAGE 33

24 influence on phytoplankton growth rates. On the other hand, maximum biomass was primarily affected by interactions between nitrogen and phosphorus, which appeared to result from responses to both the amount of nutrients and their relative abundances. The difference in minimum and maximum values for algal growth rates (d -1 ) at sites 23 and 81 were 0.09 and 0.15, respectively. These nutrient-induced changes in growth rate are similar to those reported in other studies. For example, Downing et al. (1999) reported that algal assemblages responded with changes in growth rates that ranged from 0.0 0.2 d -1 following the addition of surplus nutrients, while Elser and Frees (1995) reported changes in growth rates of 0.02-0.25 d -1 with the addition of nutrients. The range in growth rates (0.25 0.53 d -1 ) and the range in chlorophyll-a measured from the initial and final treatment subsamples (0.40-51.44 ug L -1 ) in this study are not unusual when compared to those reported for other systems. Growth rates (d -1 ) reported in the literature surveyed ranged from 0.10 0.80 (Elser and Frees 1995, Hein and Riemann 1995, Phlips et al. 2002), and Frazer et al. (2004) report chlorophyll-a values that range from 0.2 74.4 ug L -1 in estuarine systems adjacent to three counties along Floridas west coast. Characterizing the two-dimensional response surfaces created in this study both qualitatively and quantitatively, can provide valuable information in identifying ways of managing nitrogen and phosphorus input to achieve management goals. For systems where algal biomass is a primary contributor to water clarity, like Kings Bay, the maximum biomass results of this study could be used to calculate the algal biomass that could be expected given a particular nutrient reduction strategy. Currently, nutrient concentrations in Kings Bay are relatively low in comparison to other coastal, spring-fed

PAGE 34

25 systems in Florida (see Frazer et al. 2001). However, nutrient concentrations (nitrate in particular) are elevated compared to the presumed historical values (see Jones and Upchurch 1994). As point sources of nutrient load to the Kings Bay system have been removed, additional nutrient reduction will require reduction of non-point sources. This may prove to be a difficult task considering the rapid rate of population growth and the associated changes in land-use within the broader Kings Bay watershed (Jones and Upchurch 1994, SWFWMD 2003). This being the case, managers seeking to achieve an increase in water clarity in Kings Bay may have to focus on other factors that influence the accumulation of phytoplankton biomass such as removal processes. In shallow estuarine systems like Kings Bay, phytoplankton are typically removed by physical processes, e.g., flushing, and potentially by grazing (Phlips et al. 2002). Although little is known about grazing effects in Kings Bay, there have been modeling studies that characterize circulation patterns and calculate flushing rates. Open waters of the bay are thought to have relatively short particle residence times, between 50-59 hrs (2 2.5 days), while dye injection studies suggest that Kings Bay is flushed every 71-94 hrs (3 4 days), depending on the magnitude of spring discharge (Hammett et al. 1996). Ambient phytoplankton growth rates measured in this study suggest that phytoplankton can double in as little as 1.4 days. At present, phytoplankton doubling times are close to the flushing rate. It may be possible to reduce phytoplankton growth rates to the point where doubling times are greater than flushing rates, thereby potentially reducing the accumulation of phytoplankton biomass within the bay. The magnitude of nutrient limitation can be used to determine the reduction in nutrient concentration necessary to achieve the desired response in phytoplankton growth rates. For example, the overall

PAGE 35

26 magnitude of nitrogen limitation for site 81 estimates a 0.1011 change in growth rate per mg L -1 of nitrogen. To increase doubling times from 1.4 days to 2 days, phytoplankton growth rate would have to decrease from 0.4836 to 0.3466. The magnitude of nitrogen limitation suggests that site 81 would have to undergo a 1.4-mg L -1 reduction of nitrogen if flushing rates are the only factor considered to remove phytoplankton. However, the nitrogen concentration in Kings Bay is already so low (TN ~0.25 mg L -1 Hoyer et al. 1997), that a 1.4-mg L -1 reduction in nitrogen is not possible. A similar calculation with phosphorus indicates that a decrease in phytoplankton growth rate from 0.4836 to 0.3466 at site 81 would require an estimated 0.12-mg L -1 reduction of phosphorus. The average TP in Kings Bay is ~0.029 mg L -1 (Hoyer et al. 1997), thus eliminating the reduction of phosphorus as a possibility. Instead a more realistic approach to improving water clarity in Kings Bay may include a combination of nutrient remediation strategies for reducing phytoplankton as well as other means of improving water clarity such as sediment stabilization or increased water flow. An important factor to consider when using estimates like the ones calculated in this study is the spatial and temporal variation of phytoplankton responses. When making management decisions, it is not only important to understand what factors are affecting the biogeochemical processes of the system, but where and how these processes are affected. Although only two sites were used in this study, the results suggest that different areas within the same system may be controlled by different limiting factors. This phenomenon is not restricted to Kings Bay; Lake Okeechobee and Chesapeake Bay have also been found to show similar spatial heterogeneity of nutrient limitation (Aldridge et al. 1995, Fisher et al. 1999). It is important that these patterns, along with seasonal and

PAGE 36

27 annual variations are taken into consideration in order to make effective management decisions (Vanni and Temte 1990, Lewitus et al. 1998).

PAGE 37

LIST OF REFERENCES Aldridge, F. J., E. J. Phlips, and C. L. Schelske. 1995. The use of nutrient enrichment bioassays to test for spatial and temporal distribution of limiting factors affecting phytoplankton dynamics in Lake Okeechobee, Florida. Archives Hydrobiologica Special Issues on Advanced Limnology 45: 177-190. American Public Health Association (APHA). 1998. Standard methods for the examination of water and wastewater. 20 th Ed. American Public Health Association, Washington, DC. Baird, D., R. R. Christian, C. H. Peterson, and G. A. Johnson. 2004. Consequences of hypoxia on estuarine function: energy diversion from consumers to microbes. Ecological Applications 14(3): 805-822. Beardall, J., E. Young, and S. Roberts. 2001. Approaches for determining phytoplankton nutrient limitation. Aquatic Sciences 63: 44-69. Bishop, J. H., and D. E. Canfield. 1995. Volunteer water quality monitoring at Crystal River, Florida (August 1992 August 1995). Final Report. Southwest Florida Water Management District, Brooksville, FL. Carpenter, S. R., N. F. Caraco, D. L. Correll, R. W. Howarth, A. N. Sharpley, and V. H. Smith. 1998. Nonpoint pollution of surface waters with phosphorus and nitrogen. Ecological Applications 8(3): 559-568. Carpenter, S. R., D. Ludwig, and W. A. Brock. 1999. Management of eutrophication of lakes subject to potentially irreversible change. Ecological Applications 9(3): 751-771. Downing, J. A., C. W. Osenberg, O. Sarnelle. 1999. Meta-analysis of marine nutrient-enrichment experiments: variation in the magnitude of nutrient limitation. Ecology 80(4): 1157-1167. Duarte, C. M. 1992. Nutrient concentration of aquatic plants: patterns across species. Limnology and Oceanography 37(4): 882-889. Elser, J. J., and D. L. Frees. 1995. Microconsumer grazing and sources of limiting nutrients for phytoplankton growth: application and complications of a nutrient-deletion/dilution-gradient technique. Limnology and Oceanography 40(1): 1-16. 28

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29 Fisher, T. R., A. B. Gustafson, K. Sellner, R. Lacouture, L. W. Haas, R. L. Wetzel, R. Magnien, D. Everitt, B. Michaels, and R. Karrh. 1999. Spatial and temporal variation of resource limitation in Chesapeake Bay. Marine Biology 133: 763-778. Frazer, T. K., and J. A. Hale. 2001. An atlas of submersed aquatic vegetation in Kings Bay (Citrus County, Florida). Southwest Florida Water Management District, Brooksville, FL. Report 99CON000041. pp. 14. Frazer, T. K., M. V. Hoyer, S. K. Notestein, J. A. Hale, and D. E. Canfield. 2001. Physical, chemical and vegetative characteristics of five Gulf coast rivers. Final Project Report. Southwest Florida Water Management District, Brooksville, FL. Frazer, T. K., S. K. Notestein, C. A. Jacoby, and J. A. Hale. 2004. Water quality caracteristics of the nearshore gulf coast waters adjacent to Citrus, Hernando, and Levy Counties: Project COAST 1997 to 2003. Annual Report. Southwest Florida Water Management District, Brooksville, FL. Frazer, T. K., E. J. Phlips, S. K. Notestein, and C. Jett. 2002. Nutrient limiting status of phytoplankton in five gulf coast rivers and their associated estuaries. Final Report. Southwest Florida Water Management District, Brooksville, FL. Gerhart, D. Z., and G. E. Likens. 1975. Enrichment experiments for determining nutrient limitation: four methods compared. Limnology and Oceanography 20(4): 649-653. Grall, J., and L. Chauvaud. 2002. Marine eutrophication and benthos: the need for new approaches and concepts. Global Change Biology 8: 813-830. Haller, W. T., J. V. Shireman, and D. E. Canfield, Jr. 1983. Vegetation and herbicide monitoring study in Kings Bay, Crystal River, FL. U.S. Army Corps of Engineers, Jacksonville, FL. Report DACW17-80-C-0062. pp. 169. Hammett, K. M., C. R. Goodwin, and G. L. Sanders. 1996. Tidal-flow, circulation, and flushing characteristics of Kings Bay, Citrus County, Florida. U. S. Geological Survey, Tallahassee, FL. Report 96-230. pp. 63. Hauxwell, J., C. W. Osenberg, and T. K. Frazer. 2004. Conflicting management goals: manatees and invasive competitors inhibit restoration of a native macrophyte. Ecological Applications 14(2): 571-586. Hein, M., and B. Riemann. 1995. Nutrient limitation of phytoplankton biomass or growth rate: an experimental approach using marine enclosures. Journal of Experimental Marine Biology and Ecology 188: 167-180. Hoyer, M. V., L. K. Mataraza, A. B. Munson, and D. E. Canfield, Jr. 1997. Water clarity in Kings Bay/ Crystal River. Southwest Florida Water Management District, Brooksville, FL. pp.22.

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30 Jones, G. W., and S. B. Upchurch. 1994. Origin of nutrients in ground water discharging from the Kings Bay springs. Southwest Florida Water Management District, Brooksville, FL. pp. 120. Lapointe, B. E., P. J. Barile, and W. R. Matzie. 2004. Anthropogenic nutrient enrichment of seagrass and coral reef communities in the Lower Florida Keys: discrimination of local versus regional nitrogen sources. Journal of Experimental Marine Biology and Ecology 308: 23-58. Lewitus, A. J., E. T. Koepfler, and J. T. Morris. 1998. Seasonal variation in the regulation of phytoplankton by nitrogen and grazing in a salt-marsh estuary. Limnology and Oceanography 43(4): 636-646. Lund, J. W. G. 1972. Eutrophication. Proceedings of the Royal Society of London. Series B, Biological Sciences 180(1061): 371-382. Mller, R., and P. Stadelmann. 2004. Fish habitat requirements as the basis for rehabilitation of eutrophic lakes by oxygenation. Fisheries Management and Ecology 11: 251-260. Notestein, S. K., T. K. Frazer, S. R. Keller, and R. A. Swett. 2005. Kings Bay Vegetation Evaluation 2004. Southwest Florida Water Management District, Brooksville, FL. pp. 94. Osenberg, C. W., C. M. St. Mary, J. A. Wilson, and W. J. Lindberg. 2002. A quantitative framework to evaluate the attraction-production controversy. ICES Journal of Marine Science 59: S214-S221. Phlips, E. J., S. Badylak, and T. Grosskopf. 2002. Factors affecting the abundance of phytoplankton in a restricted subtropical lagoon, the Indian River Lagoon, Florida, USA. Estuarine, Coastal, and Shelf Science 55: 385-402. Redfield, A. C. 1934. On the proportions of organic derivatives in sea water and their relation to the composition of plankton. In: James Johnstone Memorial Volume. (pp. 176-192) University Press, Liverpool, UK. Statistical Analysis System (SAS) Institute. 2004. SAS users guide. SAS Institute, Inc. Cary, North Carolina. Smith, V. H., G. D. Tilman, and J. C. Nekola. 1999. Eutrophication: impacts of excess nutrient inputs on freshwater, marine, and terrestrial ecosystems. Environmental Pollution 100: 179-196. Stewart-Oaten, A. 1996. Problems in the analysis of environmental monitoring data. In Detecting ecological impacts: concepts and applications in coastal habitats, pp. 109-131. Ed. By R. J. Schmitt, and C. W. Osenberg. Academic Press, San Diego, USA. 401 pp.

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31 Southwest Florida Water Management District (SWFWMD). 2003. Crystal River/ Kings Bay Summary Report. Southwest Florida Water Management District, Brooksville, FL. pp. 33. Terrell, J. B., and D. E. Canfield, Jr. 1996. Evaluation of the effects of nutrient removal and the Storm of the Century on submersed vegetation in Kings Bay Crystal River, Florida. Journal of Lake and Reservoir Management 12(3): 394-403. Tett, P., L. Gilpin, H. Svendsen, C. P. Erlandsson, U. Larsson, S. Kratzer, E. Fouilland, C. Janzen, J. Lee, C. Grenz, A. Newton, J. G. Ferreira, T. Fernandes, and S. Scory. 2003. Eutrophication and some European waters of restricted exchange. Continental Shelf Research 23: 1635-1671. United States Fish and Wildlife Service (USFWS). 2005. Crystal River National Wildlife Refuge. http://crystalriver.fws.gov/ Last assessed August 2005. Vanni, M. J., and J. Temte. 1990. Seasonal patterns of grazing and nutrient limitation of phytoplankton in a eutrophic lake. Limnology and Oceanography 35(3): 697-709. Yobbi, D. K., and L. A. Knochenmus. 1989. Effects of river discharge and high-tide stage on salinity intrusion in the Weeki Wachee, Crystal, and Withlacoochee river estuaries, southwest Florida. U. S. Geological Survey, Tallahassee, Florida. Report 88-4116. pp.63.

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BIOGRAPHICAL SKETCH I am originally from St. Louis, MO. I moved to Florida to get my B.S. in marine science at Eckerd College in St. Petersburg, FL. For my undergraduate thesis, I studied the historic spatial and temporal changes in submerged aquatic vegetation in Tampa Bay, FL, using aerial photography. After graduating, I spent a year working in the chemistry lab at the Florida Wildlife Research Institute in St. Petersburg, FL. I then came to the University of Florida to earn my M.S. under the advisement of Dr. Tom Frazer at the Department of Fisheries and Aquatic Sciences. 32


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Title: Quantifying the Magnitude of Nutrient Limitation on Phytoplankton in Kings Bay, Florida, USA
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Material Information

Title: Quantifying the Magnitude of Nutrient Limitation on Phytoplankton in Kings Bay, Florida, USA
Physical Description: Mixed Material
Copyright Date: 2008

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QUANTIFYING THE MAGNITUDE OF NUTRIENT LIMITATION ON
PHYTOPLANKTON IN KINGS BAY, FLORIDA, USA















By

DARLENE SAINDON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Darlene Saindon

































This document is dedicated to my family: Mike, Jeannie, Anna, and Travina, and to the
past, present, and future generations of staff and graduate students of the Frazer Lab at
the University of Florida, especially Steph, Sky, Kelly, Kristin, Vince, Emily, and Ray.















ACKNOWLEDGEMENTS

I thank my committee members for their support and guidance. I also thank the

students and staff of the University of Florida's Department of Fisheries and Aquatic

Science for use of their equipment and their assistance in running this experiment.

Funding for this project was provided by the University of Florida's Institute of Food and

Agricultural Science and the Swisher Water Resources Graduate Research Assistantship.
















TABLE OF CONTENTS



A C K N O W L E D G E M E N T S ..................................................................... .................... iv

LIST OF TABLES ............... .................................... vi

LIST OF FIGURE S ......... ..................................... ........... vii

A B S T R A C T .......................................... .................................................. v iii

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 ST U D Y L O C A T IO N ................................................................................ ............5

3 M E T H O D S ............................................................................. 7

Water Collection...............................7.... .........7
Laboratory Assays of Nutrient Limitation......... ................... ......... ...................7
C hlorophyll A naly sis .. .. ...... ............................................................ ...... ....... .. ..
N utrient Analysis ................................................................ ........ 10
Calculation of Growth Rates and Maximum Biomass ........................................... 10
Statistical A n aly sis ................................................. ...................10
N utrient Lim itation A ssessm ent ....................................................... ..... ........... 11

4 RESULTS ..................................... .................................. ........... 13

A m bient C condition s ................................................................ .. ...... ... ...... 13
Experim mental A ssay .................. ..................................... .. .......... .... 14

5 DISCUSSION .................. .................................... ........... .... .......... 23

Comparing Ambient and Experimental Results ............................... ................ 23
E xperim ental A ssay Im plications.................................................................... .......23

L IST O F R E FE R E N C E S ............................................................................. .............. 28

B IO G R A PH IC A L SK E TCH ..................................................................... ..................32




v
















LIST OF TABLES


Table p

3-1 Experimental design with bioavailable N:P ratios at the start of the assays for
water collected from sites 23 (a) and 81(b)....................................................... 12

4-1 Summary of ambient water quality parameters for sites 23 and 81 ...........................16

4-2 Summary of statistical results for the overall effects of nitrogen and phosphorus on
phytoplankton growth rates and maximum biomass........................................17

4-3 Summary of statistical results for the effects of nitrogen and phosphorus on
phytoplankton maximum biomass at fixed levels of the other nutrient................... 18
















LIST OF FIGURES

Figure page

2-1 M ap of K ings B ay ................ .................................... ...... ........ ... .. .. ..6

4-1 Two dimensional growth rate response surface for site 23 .............. ..................19

4-2 Two dimensional growth rate response surface for site 81 ......................................20

4-3 Two dimensional maximum biomass response surface for site 23 ...........................21

4-4 Two dimensional maximum biomass response surface for site 81 ............................22















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

QUANTIFYING THE MAGNITUDE OF NUTRIENT LIMITATION ON
PHYTOPLANKTON IN KINGS BAY, FLORIDA, USA

By

Darlene Saindon

December 2005

Chair: Tom Frazer
Co-Chair: Craig Osenberg
Major Department: Fisheries and Aquatic Sciences

The recreational and economic viability of many aquatic systems is linked to water

quality in general and water clarity in particular. In cases where water clarity has been

impaired as a consequence of excessive phytoplankton production, a common

management strategy is to reduce the load of the limiting nutrient. In this study, nutrient

addition and dilution assays were used to quantify the magnitude of nutrient (nitrogen

and phosphorus) limitation, and characterize the two-dimensional response surface of

phytoplankton across a broad range of nutrient concentrations and N:P ratios. Site water

from two sites in Kings Bay, Florida (USA) was filtered using a Millipore stirred cell

concentrator, which allowed a fixed percentage of site water and nutrients to be removed

while maintaining ambient abundances of plankton. DI water and stock solutions of

nitrogen and phosphorus were added to return samples to their original volumes and

create nutrient concentration treatments that ranged from -12 to 10 times the ambient

concentration of phosphorus, and 1/4 to 15 times the ambient concentration of nitrogen.









Growth rates and maximum biomass were estimated using in vivo fluorescence measures.

The magnitude of limitation was quantified by estimating how much the response

variable changed per unit of nutrient concentration. Phytoplankton growth rates were not

limited by nitrogen or phosphorus at one site, but limited by both nitrogen and

phosphorus at the other site. The overall estimated magnitude of limitation for this latter

site was a 0.1011 change in per capital algal growth (d-1) for each mg L-1 of nitrogen, and

a 1.1268 change in per capital algal growth (d-1) for each mg L-1 of phosphorus. Neither

site showed significant nitrogen/phosphorus interaction effects on growth rate responses.

However, there was a significant interactive effect of nitrogen and phosphorus on

maximum biomass responses at both sites. A qualitative assessment of the response

surfaces for maximum biomass suggests that this variable is affected by both the N:P

ratio of nutrient treatments as well as the total amount of nutrients supplied. Since the

magnitude of limitation of one nutrient is determined by the concentration of the other

nutrient, an overall magnitude of limitation may either overestimate or underestimate the

expected maximum biomass response for specific co-occurring changes in nutrient

concentrations. Instead, magnitude of limitation should be determined on a case by case

basis, taking into consideration specific nutrient remediation scenarios and creating a

response surface with those co-occurring changes in nutrient concentrations in mind. The

combination of quantitative analyses and characterization of two-dimensional response

surfaces can be used by water resource managers as a tool to develop and implement

nutrient reduction strategies with predictable outcomes.














CHAPTER 1
INTRODUCTION

Eutrophication of aquatic ecosystems, as a consequence of increased nutrient

delivery, occurs worldwide. It can result in an increase in the frequency and intensity of

algal blooms and fish kills, as well as a decrease in biodiversity with losses of key species

such as rooted aquatic plants and corals (Grall and Chauvaud 2002, Baird et al. 2004,

Hauxwell et al. 2004, Lapointe et al. 2004, Miller and Stadelmann 2004). The resulting

decline in water quality and ecosystem function can have pronounced socioeconomic

impacts that negatively affect a large number of recreational, agricultural, and industrial

interests (Lund 1972, Carpenter et al. 1998, Smith et al. 1999, Tett et al. 2003). In

response to the many issues related to cultural eutrophication, there has been a concerted

effort on the part of scientists and water resource managers to improve water clarity and

reverse the effects of eutrophication. Nutrient reduction strategies, however, have

resulted in only limited success. In fact, many systems show no response to nutrient

reduction. Carpenter et al. (1999), for example, showed that the effects of prior nutrient

enrichment could be (1) reversible (recovery is immediate and proportional to the nutrient

reduction), (2) hysteretic (recovery requires extreme reductions in nutrient input for a

period of time), or (3) irreversible (recovery cannot be accomplished by reducing nutrient

input alone). To determine why hysteretic and irreversible systems do not respond to

nutrient remediation and reduction strategies, scientists and managers are now focused on

better understanding the complex suite of physical, chemical, and biological interactions

that affect nutrient uptake and assimilation.









An understanding of the relationship between phytoplankton abundance and

nutrient availability is vital to our ability to predict the effects of either nutrient increases

or reductions. This relationship is especially important for systems where phytoplankton

are one of the primary factors reducing water clarity. Of particular importance is the

ability to determine whether or not phytoplankton growth is limited by a particular

nutrient and identifying what that nutrient is. Nutrient limitation may be inferred from an

analysis of nutrient availability and elemental ratios in phytoplankton or bulk suspended

material in the water column. A more direct experimental evaluation can be made by

adding nutrients to a water sample and evaluating if the growth, biomass, or other

physiological parameter (e.g., respiration or carbon fixation rate) of phytoplankton differs

significantly from a control treatment (Gerhart and Likens 1975, Beardall et al. 2001).

Results from these types of studies can yield important insights to the current limiting

status of a system, but may be of limited utility to managers that need to make

quantitative predictions of a system's behavior to specific management actions.

There are three primary limitations of this simple experimental approach. First, it

focuses on two nutrient concentrations (added and control), which may or may not be

relevant to the situations of interest to the managers. Second, many studies are not able to

capture any complex interactions that may be occurring among nutrients. Some studies

focus on the manipulation of a single nutrient, whereas others have limited nutrient

treatment combinations (2x2 factorial with added and control treatments for each

nutrient). In either case, interactions among nutrients can not be characterized. By

manipulating >1 nutrient, all at a range of concentrations, multi-dimensional response

surfaces can be obtained and characterized to reveal potentially complex nutrient









interactions. Finally, the standard experimental approach focuses on the detection of

statistical differences between treatments, which may not reveal biologically important

effects which may or may not be statistically significant (e.g., Stewart-Oaten 1996).

Instead, quantitative estimates of the magnitude of limitation (e.g., Downing et al. 1999,

and Osenberg et al. 2002) may be more useful because these estimates reveal how much

systems might respond to changes in nutrients. As a result, scientists and managers can

directly test responses of phytoplankton to the range of nutrient concentrations they are

currently exposed to or may be exposed to in the future. This information may provide

managers with the detailed information necessary to implement policies that have an

increased probability of success.

This study introduces a method combining nutrient addition and dilution assays to

create a two-dimensional response surface, and thus quantify the magnitude of nitrogen

and phosphorus limitation. Both phytoplankton growth rates and maximum achieved

biomass were measured to better understand how phytoplankton might respond to

changes in nitrogen and phosphorus concentrations. Conceptually, there are four possible

qualitative outcomes of this experiment based on the two response variables: (1) nutrients

are never limiting, i.e. initial growth rates and maximum achieved biomass are similar

across all nutrient treatments, (2) nutrients are not limiting at first, but become limiting as

nutrients are depleted, i.e. initial growth rates are similar across treatments, but maximum

achieved biomass increases with increasing nutrient concentration, (3) the nutrient is

limiting at first, but some other factor consistent across treatments becomes limiting

when the culture reaches a particular biomass, i.e. initial growth rates increase with

increasing nutrient concentration, but maximum achieved biomass are similar across all






4


nutrient treatments, and (4) the nutrient is always limiting; i.e., both initial growth rates

and maximum achieved biomass increase with increasing nutrient concentration. The

potential effects of nutrient manipulation on phytoplankton growth and biomass have

seldom been quantified. Thus, the methodology and results reported herein will be of

interest to a broad group of scientists and water resource managers.














CHAPTER 2
STUDY LOCATION

Kings Bay (Figure 2-1) is a tidally influenced system (ca. 1.75 km2; see Haller et

al. 1983) that is connected to the Gulf of Mexico by Crystal River on the west coast of

Florida, about 96-km north of Tampa (Frazer et al. 2001). Greater than 30 springs in

Kings Bay have a combined average total discharge of -27 m3 s-1 accounting for

approximately ninety-nine percent of the freshwater entering the system (Yobbi and

Knochenmus 1989, Hammett et al. 1996). Over 100,000 boaters, anglers, and sport divers

visit these springs each year (USFWS 2005). Kings Bay is within the confines of the

Crystal River National Wildlife Refuge and provides valuable habitat for a variety of

native and endangered species. Since 1999, greater than 250 West Indian manatees

(Trichechus manatus) take refuge annually in the relatively warm spring waters during

winter months (J. Kleen, USFWS, pers. comm.). This system is a recreationally,

economically, and ecologically important area that relies on maintaining water clarity and

functional habitats to continue being utilized in this manner.

As part of the Surface Water Improvement and Management (SWIM) program, one

of the primary management goals for Kings Bay is to improve water clarity and wildlife

habitat (SWFWMD 2003). Hoyer et al. (1997) found that the water clarity in Kings Bay

is primarily determined by algal suspended particles, and moderately affected by non-

volatile and detrital suspended particles. In an effort to reduce eutrophication, point

sources of nutrients (e.g. effluent from the City of Crystal River Sewage Treatment Plant)

were removed in 1992. As a result, average total phosphorus was reduced from 105 ug









L-1 to 27 ug L-1 and average total nitrogen was reduced from 620 ug L-1 to 220 ug L-1 in

Cedar Cove located in the northern region of Kings Bay (Terrell and Canfield 1996).

Water clarity, however, has not improved (Bishop and Canfield 1995, Hoyer et al. 1997,

SWFWMD 2003).


0 0.5 1 Kilometers b1 f.l

Figure 2-1 Map of Kings Bay with sampling sites. There are > 30 springs located
throughout Kings Bay with the main spring for Kings Bay located in the
southern region, southeast of Site 81. Prior to 1992, the Crystal River Sewage
Treatment Plant discharged effluent into Cedar Cove in the northeast region of
Kings Bay. The dark grey/ black areas surrounding the bay correspond to
developed coastline, whereas the light grey areas correspond to marshland.














CHAPTER 3
METHODS

Water Collection

Two sites within Kings Bay were selected for water collection and subsequent

nutrient manipulation. These two sites, numbers 23 and 81 (see Figure 2-1), correspond

geographically to those established by Frazer and Hale (2001) as part of a longer-term

vegetative monitoring program (see Notestein et al. 2005). On October 11, 2004, surface

water samples (- 0.5 m depth) from both sites were collected and analyzed within 24h for

nitrate + nitrite (N03- + NO2-) and soluble reactive phosphorus (SRP) to determine

ambient concentrations. These values were then used to estimate the amount of stock

solutions needed to create designated treatments (see Table 3-1). The next day, water was

again collected at each site (20-L samples). Water samples were transported immediately,

in covered carboys, to the laboratory and allowed to set in the dark overnight in an

incubation room before initiation of the experiment the following morning.

Laboratory Assays of Nutrient Limitation

Phytoplankton growth rate (d-1) and maximum algal biomass, as indicated by in

vivo fluorescence (IVF) measures of chlorophyll-a, were determined over a gradient of

nitrogen and phosphorus concentrations. The experiment was set up as a fully crossed

factorial design, with 5 manipulated nitrogen and 5 manipulated phosphorus treatments.

Each treatment was replicated three times. Target phosphorus treatments corresponded

approximately to ambient SRP (X), 50% dilution (0.5 X), 25% dilution (0.75 X), 25%

addition (1.25 X), and 50% addition (1.5 X). Nitrogen treatments were chosen to









correspond to ambient nitrate + nitrite (X), 50% dilution (0.5 X), and additions that were

double (2 X), four times (4 X), and eight times (8 X) ambient, reflecting a particular

interest in and concern for increased nitrate in this and other spring-fed systems in Florida

(SWFWMD 2003). An additional control (run in triplicate) that lacked a nutrient

manipulation was used to determine ambient conditions and verify that the responses of

phytoplankton to experimental nutrient manipulation were realistic. Because the target

nutrient treatment levels were chosen based on water analyses run the previous day, the

ambient nutrient levels ("X") were not necessarily identical to the nutrient levels in the

ambient control treatment.

To establish the nutrient gradients, water collected from the two sites was filtered

using a Millipore stirred cell concentrator (model 8400), with Biomax-30 filters

(NMWL: 30,000). This allowed a pre-determined percentage of site water and nutrients

to be removed while maintaining ambient abundances of plankton. For all replicates

within a treatment, except the ambient control, a 400-ml aliquot of site water was filtered

to 50% of its original volume to control for filtration effects. The unfiltered portion, with

ambient concentration of plankton, was then placed in a 500-ml Erlenmeyer flask. DI

water and stock solutions of known nitrogen (KNO3) and phosphorus (K2HPO4)

concentration were added to return samples to the original volume of 400 ml and

establish the nutrient concentration treatments (see Table 3-1). For the ambient control,

site water was filtered to 50% of the original volume to control for filtration effects, and

the filtrate, rather than DI water, added back to the sample to maintain both ambient

nutrient and plankton concentrations. This control was used to determine ambient growth

rate and biomass, and provided a comparison to determine if there was an effect of









removing micronutrients from the experimental assays. Within 5 min of the nutrients

being added, 100 ml of sample water from each flask was withdrawn and later analyzed

for initial chlorophyll-a and nutrient concentrations. Nutrient measurements include total

nitrogen (TN), nitrate + nitrite (NO3- +NO2-), ammonium (NH4+), total phosphorus (TP),

and soluble reactive phosphorus (SRP). The nitrogen and phosphorus treatments reported

herein are the averaged initial nitrate + nitrite and SRP concentrations measured from the

assays.

All Erlenmeyer flasks were subsequently incubated in temperature-controlled water

baths with bottom illumination (-120 Em-2s-1). Incubation temperatures were maintained

at ambient temperatures (24C) recorded on the water collection date with a photoperiod

of 11/13 dark/light hours. Algal biomass was estimated using IVF measures at the time of

nutrient addition (time 0), and at 12 h intervals thereafter until IVF values in all

experimental flasks had peaked. IVF was measured using a Turner Designs Model 10

fluorometer with a 1-cm path length. At the end of the incubation period, water remaining

in the flasks was used for final IVF, chlorophyll-a, and nutrient measurements.

Chlorophyll Analysis

Initial and final chlorophyll-a measurements were obtained by filtering a minimum

of 50 ml of sample water through a 47-mm Whatman GF/F filter. Filters were stored

over desiccant and frozen prior to analysis. Chlorophyll-a was extracted in a 90% heated

ethanol solution, and chlorophyll-a concentrations were determined

spectrophotometrically using a Digilab Hitachi U-2810 spectrophotometer with a 4-cm

path length (APHA 1998).









Nutrient Analysis

Total nitrogen and total phosphorus concentrations were determined from whole

water samples digested with potassium persulfate. Ammonium, nitrate + nitrite, and SRP

were determined from water samples filtered through a 47-mm Whatman GF/F filter.

Total nitrogen, ammonium, and nitrate + nitrite analyses were determined

spectrophotometrically on a Bran-Luebbe autoanalyzer with a cadmium column

reduction method. Total phosphorus and SRP were determined spectrophotometrically on

a Digilab Hitachi U-2810 spectrophotometer with a 4-cm path length (APHA 1998).

Calculation of Growth Rates and Maximum Biomass

Phytoplankton growth rates (d-1) were estimated as ln(IVFt/IVFo)/t, where IVFo was

the initial fluorescence and IVFt was the fluorescence after t days, where t was

determined visually as the time period during which In(IVF) was an approximately linear

function of time. Maximum biomass was determined as the peak fluorescence measured

just before IVF began to decrease as the algal assemblage crashed. This peak sometimes

coincided with the end of exponential growth, but usually lagged by 0.5-1.5 days.

Statistical Analysis

Because nitrogen and phosphorus treatments were not set at the same

concentrations for the two sites, an analysis of co-variance (ANCOVA, with [N] and [P]

as the covariates) was used to determine whether the two sites showed similar responses

to nutrient treatments. An analysis of variance (ANOVA) was then run for each site

individually to determine how nutrient treatments affected phytoplankton growth rates

and maximum biomass. For all analyses, the simplest model was used by removing

higher order treatment effects that were not significant; nutrient effects were determined

to be significant at a = 0.05 (SAS Institute 2004).









Nutrient Limitation Assessment

The magnitude of nutrient limitation is reflected as the change of the response

variable (i.e. growth rate or maximum biomass) for a given unit change of nitrogen or

phosphorus concentration. Graphically, the magnitude of limitation is the slope of the

relationship between growth rate (or maximum biomass) and nutrient concentration. If

there is no interaction between nitrogen and phosphorus, then the magnitude of limitation

for one nutrient can be expressed independently of the concentration of the other nutrient.

Where there was no nitrogen/phosphorus interactions, estimates of the treatment effects

determined by regression analysis were used as the overall estimate of magnitude of

limitation. If there is an interaction between N and P, then the magnitude of limitation for

one nutrient is influenced by the concentration of the other nutrient. Where there were

nitrogen/phosphorus interactions, estimates of treatment effects were also determined

using regression analysis for nutrients at each concentration level of the other nutrient.










Bioavailable N:P ratios at the start of the assays for water collected from sites
23 (a) and 81 (b). N:P ratios < 7 (shaded) suggest nitrogen limited conditions;
N:P ratios > 7 unshadedd) suggest phosphorus limited conditions. Nitrogen
and phosphorus treatments reported are the averaged beginning nitrate +
nitrite and SRP concentrations for each target treatment (n=15). Standard
deviations are given in parentheses and the experimental treatments closest to
ambient nutrient concentrations for each site are indicated with an asterisk.


Table 3-1


(a) Site 23 Nitrogen (ug/L)

25 65 167 385 794
(7.0) (2.3) (11.1) (9.6) (20.6)

7(1.6) 3.57 9.29 23.86 55.00 113.43


CM 12(1.1) 2.08 *5.42 13.92 32.08 66.17
U)
18 (1.0) 1.39 3.61 9.28 21.39 44.11
0

0 25(1.0) 1.00 2.60 6.68 15.40 31.76


32 (1.1) 0.78 2.03 5.22 12.03 24.81


(b) Site 81 Nitrogen (ug/L)

7 83 191 393 802
(3.8) (3.6) (8.1) (23.3) (42.4)

4 (1.4) 1.75 20.75 47.75 98.25 200.50


CM 9(1.2) 0.78 *9.22 21.22 43.67 89.11

0
16(1.1) 0.44 5.19 11.94 24.56 50.13

0 23 (1.1) 0.30 3.61 8.30 17.09 34.87

30 (1.1) 0.23 2.77 6.37 13.10 26.73














CHAPTER 4
RESULTS

Ambient Conditions

Sites 23 and 81 had similar temperature, salinity, dissolved oxygen, and pH, but

different nutrient concentrations (see Table 4-1). Ambient nitrate + nitrite and SRP

concentrations at site 23 (83 ug L-1 and 11 ug L-1, respectively) were approximately twice

those at site 81 (39 ug L-1 and 6 ug L-1, respectively). The bioavailable N:P ratio (ug:ug;

nitrate + nitrite:SRP) at site 23 was 7.6, while at site 81 the N:P ratio was 6.5. Both sites

23 and 81 were near the expected 7:1 N:P weight ratio for the chemical composition of

phytoplankton (Redfield 1934, Duarte 1992). This 7:1 N:P ratio is generally considered

the threshold where phytoplankton switch from nitrogen limitation (N:P < 7) to

phosphorus limitation (N:P > 7). Qualitative assessment of the plankton community

suggests that both sites were comprised predominately of small centric diatoms and

cryptophytes. There was, however, a lower concentration of chlorophyll-a at site 23

(10.92 ug L-) than at site 81 (14.59 ug L-1).

By using the model created by the experimental treatments, the ambient control

responses can be compared to the experimental treatment responses to determine if the

methodology may have affected the results. The maximum biomass achieved in the

ambient control assays were 0.8023 for site 23 and 0.6117 for site 81, as indicated by IVF

measures. By using the regression model created from the experimental assays (see Table

4-2), the maximum biomass (1SE) for site 23 was expected to be 0.7042 0.1118, and

site 81 was expected to be 0.7121 0.1187. Since the actual maximum biomass









measured in the ambient control assays fall within these ranges, the methodology did not

appear to bias the maximum biomass results. On the other hand, ambient growth rates for

phytoplankton at sites 23 and 81 were 0.3494 and 0.4836 d-1, respectively. Again, by

using the regression model created from the experimental assays (see Table 4-2), the

growth rate (1SE) for site 23 was expected to be 0.3019 0.0300, and site 81 was

expected to be 0.4318 0.0400. Growth rates measured in the ambient control assays

were greater than the expected, suggesting that the methodology employed may have

slightly biased the growth rate results.

Experimental Assay

Mean algal growth rates in the assays ranged from 0.2493 to 0.3406 d-1 for site 23,

and 0.3826 to 0.5349 d-1 for site 81 (Figures 4-1 and 4-2, respectively). The mean

maximum biomass achieved across treatments, as indicated by IVF measures, ranged

from 0.5667 to 1.0411 for site 23, and 0.6167 to 1.0179 for site 81 (Figures 4-3 and 4-4,

respectively). There was a significant site effect on growth rate (F2, 142 = 939.47, P <

0.0001) and maximum biomass (F2, 138 = 161.89, P < 0.0001). In addition, there was a

significant site and nitrogen treatment interaction effect (F1, 142= 5.18, P = 0.0243) on

growth rate, as well as significant nitrogen x phosphorus (F1, 138 = 13.99, P = 0.0003) and

nitrogen2 x phosphorus (F1, 138= 12.53, P = 0.0005) effects on maximum biomass. As a

consequence of the significant site effects, phytoplankton responses to nutrient treatments

were assessed for each site individually.

Nitrogen and phosphorus had significant effects on phytoplankton growth rates

only at site 81 (Table 4-2; Figures 4-1 and 4-2). The overall magnitude of nitrogen

limitation (+1SE) at this site was estimated as 0.1011 0.0160 L mg-1 d-1 (i.e. a 10%

increase in growth for each 1 mg L-1 increase in nitrogen concentration). Magnitude of









limitation for phosphorus (1SE), at site 81, was estimated as 1.1268 0.4866 L mg-' d-.

For site 23, the estimated magnitude of limitation (1SE) by both nitrogen (-0.0112 +

0.0120) and phosphorus (-0.1899 0.3792) included zero; hence the absence of a

significant effect of the nitrogen or phosphorus gradient.

With regard to the accumulation of phytoplankton biomass (Figures 4-3 and 4-4),

there was a significant interaction between nitrogen and phosphorus on the maximum

biomass response at both sites (see Table 4-2). Due to this interaction, estimates of the

treatment effect were determined for each nutrient at fixed levels of the other nutrient

(see Table 4-3). Treatment effects of phosphorus at fixed levels of nitrogen ranged from

-0.9333 to 9.9987 (IVF) L mg-1 for site 23, and -2.3460 to 10.2664 (IVF) L mg-1 for site

81. Treatment effects of nitrogen at fixed levels of phosphorus ranged from 0.4786 to

2.0827 (IVF) L mg-1 for site 23, and 0.2339 to 1.6421 (IVF) L mg-1 for site 81. These

results are consistent with the potential role of both nitrogen and phosphorus on

phytoplankton responses as indicated with the growth rate results above.

Qualitative assessment of the response surfaces for maximum biomass suggests

that this variable is affected by both the N:P ratio of nutrient treatments as well as the

total amount of nutrients supplied. Maximum biomass appeared to be maintained at low

concentrations when the N:P weight ratios were either very low (N:P < 3) or very high

(N:P > 27). At the lowest concentrations of each nutrient (i.e. 7 ug L-1 phosphorus and 25

ug L-~ nitrogen for site 23; 4 ug L-U phosphorus and 7 ug L-U nitrogen for site 81),

maximum biomass does not significantly change regardless of the concentration of the

other nutrient. When the N:P weight ratios were between 3 and 27, maximum biomass

appeared to increase with increasing nutrient availability.










Table 4-1 Summary of ambient water quality parameters for sites 23 and 81.
Temperature, salinity, dissolved oxygen, and pH were measured in the field at
the time of collection. All other parameters were taken from a subsample of
the ambient control treatments.


Site 23


Site 81


Temperature (C) 24.18 24.15
Salinity (ppt) 0.81 0.69
Dissolved Oxygen (mg L1) 6.3 6.99
pH 8.19 8.18
Total Nitrogen (ug L1) 183 147
Nitrate + Nitrite (ug L') 83 39
Ammonia (ug L"') 7 13
Total Phosphorus (ug L1) 27 27
SRP (ug L'1) 11 6
Chlorophyll-a (ug L1) 10.92 14.59
centric centric
Predominant Species diat dit
diatoms, diatoms,
Composition cryptophytes cryptophytes
Maximum Biomass (IVF) 0.8023 0.6117
Growth Rate (d-1) 0.3494 0.4836
Doubling rate (d) 2.0 1.4










Table 4-2 Factorial ANOVA results for sites 23 and 81 including estimates and standard
error (SE) of the treatment effects. Treatment effects are considered
significant at a < 0.05 (shaded). The simplest model was used for each
analysis by removing non-significant higher order treatment interactions. The
model for growth includes nitrogen (N) and phosphorus (P) treatment effects.
The model for maximum biomass also includes an interaction of nitrogen and
phosphorus (N*P), a quadratic term for nitrogen (N*N), and an interaction of
that quadratic term with phosphorus (N*N*P).
GROWTH
Treatment Effect Estimate of Effect SE F1, 72-value P-value
Site 23 N -0.0112 0.0120 0.88 0.3518
P -0.1899 0.3792 0.25 0.6180
Treatment Effect Estimate of Effect SE F1, 72-value P-value
Site 81 N 0.1011 0.0160 40.05 < 0.0001
P 1.1268 0.4866 5.36 0.0234
MAXIMUM BIOMASS
Treatment Effect Estimate of Effect SE F1, 69-value P-value
N 0.2088 0.4470 0.22 0.6419
Site 23 N*N -0.0898 0.5256 0.03 0.8648
P -3.8406 2.7967 1.89 0.1741
N*P 56.7800 21.4759 6.99 0.0101
N*N*P -64.8845 25.2536 6.60 0.0124
Treatment Effect Estimate of Effect SE F1, 69-value P-value
N 0.1025 0.3821 0.07 0.7894
Site 81 N*N -0.1015 0.4461 0.05 0.8206
P -2.3749 2.7789 0.73 0.3957
N*P 54.0362 20.2441 7.12 0.0095
N*N*P -58.0074 23.6320 6.03 0.0166










Table 4-3 ANOVA regression analysis results for sites 23 and 81 including estimates and
standard error (SE) of the treatment effects of one nutrient at fixed levels of
the other nutrient. Treatment effects are considered significant at ca 0.05
(shaded). Treatment effects include nitrogen (N) and the quadratic term for
nitrogen (N*N) at fixed phosphorus levels (ug L-1); and phosphorus (P)
treatment effects at fixed nitrogen levels (ug L1).
SITE 23
Phosphorus Treatment Effect Estimate of Effect SE F1,s1-value P-value
7 N 0.4786 0.3469 1.9 0.1928
N*N -0.3931 0.4079 0.93 0.3542
12 N 0.954 0.3195 8.92 0.0114
N*N -0.8705 0.3757 5.37 0.0390
18 N 1.4974 0.4520 10.97 0.0062
N*N -1.701 0.5315 10.24 0.0076
25 N 1.3684 0.5300 6.67 0.0240
N*N -1.3589 0.6232 4.75 0.0499
32 N 2.0827 0.5059 16.95 0.0014
N*N -2.2249 0.5949 13.99 0.0028
Nitrogen Treatment Effect Estimate of Effect SE F1,s1-value P-value
25 P 1.9124 1.3931 1.88 0.193
65 P -4.9333 1.8949 6.78 0.0219
167 P 2.6687 3.3061 0.65 0.4341
385 P 9.9987 5.2922 3.57 0.0814
794 P 0.0392 2.1218 0.00 0.9856
SITE 81
Phosphorus Treatment Effect Estimate of Effect SE F1,a1-value P-value
4 N 0.2518 0.2260 1.24 0.287
N*N -0.2660 0.2638 1.02 0.3332
9 N 0.2339 0.3223 0.53 0.482
N*N -0.0613 0.3762 0.03 0.8733
16 N 1.7768 0.3537 25.23 0.0003
N*N -2.1123 0.4129 26.17 0.0003
23 N 1.0387 0.4317 5.79 0.0331
N*N -1.2083 0.5040 5.75 0.0337
30 N 1.6421 0.5455 9.06 0.0109
N*N -1.6164 0.6368 6.44 0.026
Nitrogen Treatment Effect Estimate of Effect SE F113-value P-value
7 P -2.346 1.2318 3.63 0.0792
83 P -0.2692 3 0.01 0.9227
191 P 10.2664 2.9691 11.96 0.0042
393 P 7.428 4.5433 2.67 0.126
802 P 4.0152 3.8804 1.07 0.3197










0.6-

0.5


Growth Rate
(d-1)


0.4 -

0.3 -

0.2


0.1

0.4
7

Phosphorus
(ug L-1)


32
25


Nitrogen
(ug L-1)


Figure 4-1 Growth rates for site 23 at given phosphorus and nitrogen treatments (SE +
0.0300). There were no significant nitrogen or phosphorus effects on
phytoplankton growth rates (see Table 3-2). Colors represent growth rates
within the indicated interval and are not intended to imply statistically
significant differences.










0.6

0.5

0.4
Growth Rate
0.3
(d"1)
0.2

0.1


4
16 M Nitrogen
Phosphorus 23 30 ogen
(ug L)ug L

Figure 4-2 Growth rates for site 81 at given phosphorus and nitrogen treatments (SE +
0.0400). There was a significant nitrogen (F1, 72 = 40.05, P < 0.0001), and
phosphorus (Fi, 72= 5.36, P = 0.0234) effect on phytoplankton growth rates
(Table 3-2). Magnitude of limitation was estimated as a 0.1011 change in
growth rate (d-1) for each mg L-1 change in nitrogen, and an estimated 1.1268
change in growth rate (d-1) for each mg L-1 change in phosphorus. Colors
represent growth rates within the indicated interval and are not intended to
imply statistically significant differences.
















0.4L


0.2
0-4
7
Phosphorus
(ug L-1)


32 65
25


794
Nitrogen
(ug L-)


Figure 4-3 Maximum biomass, as indicated by in vivo fluorescence (IVF), for site 23 at
given phosphorus and nitrogen treatments (SE + 0.1118). There was a
significant nitrogen x phosphorus interaction effect (Fl, 69 = 6.99, P = 0.0101)
as well as a significant nitrogen2 x phosphorus interaction effect (Fl, 69 = 6.60,
P = 0.0124; Table 3-2). Colors represent maximum biomass within the
indicated interval and are not intended to imply statistically significant
differences.


Maximum
Biomass
(IVF)


L77










1.2



0.8
Maximum
Biomass 0.6
(IVF) 0.4
0.4

0.2


0
162 c Nitrogen
Phosphorus 23 0 rogen
(ug L1) (ug L1)

Figure 4-4 Maximum biomass, as indicated by in vivo fluorescence for site 81 at given
phosphorus and nitrogen treatments (SE + 0.1187). There was a significant
nitrogen x phosphorus interaction effect (Fl, 69 = 7.12, P = 0.0095) as well as a
significant nitrogen2 x phosphorus interaction effect (F1, 69 = 6.03, P = 0.0166;
Table 3-2). Colors represent maximum biomass within the indicated interval
and are not intended to imply statistically significant differences.
















CHAPTER 5
DISCUSSION

Comparing Ambient and Experimental Results

Maximum biomass did not appear to be affected by the methodology used in this

study, the actual maximum biomass values measured in the ambient control were within

the ranges predicted by the models created from the experimental assays. There was,

however, a discrepancy between the ambient control and the expected values from the

experimental assay models for growth rate responses; the models underestimated the

actual responses of phytoplankton growth rates for both sites. This discrepancy may be

due to the removal of micronutrients during the filtration process. Further

experimentation would have to be done to evaluate this hypothesis and determine what

other nutrients, if any, limit phytoplankton growth rates in Kings Bay.

Experimental Assay Implications

The results of this study provide important insights into how phytoplankton growth

rates and biomass are affected by nitrogen and phosphorus in Kings Bay. Phytoplankton

growth rates were not necessarily directly correlated with maximum biomass, i.e. growth

rate did not determine the maximum achieved biomass, just how long it took to reach that

biomass. The phytoplankton growth rate responses were spatially heterogeneous. Growth

rates were either affected by both nitrogen and phosphorus (site 81) or were not affected

by nitrogen or phosphorus (site 23). In addition, there was no significant

nitrogen/phosphorus interaction, indicating that the N:P ratio did not have a strong









influence on phytoplankton growth rates. On the other hand, maximum biomass was

primarily affected by interactions between nitrogen and phosphorus, which appeared to

result from responses to both the amount of nutrients and their relative abundances.

The difference in minimum and maximum values for algal growth rates (d-1) at

sites 23 and 81 were 0.09 and 0.15, respectively. These nutrient-induced changes in

growth rate are similar to those reported in other studies. For example, Downing et al.

(1999) reported that algal assemblages responded with changes in growth rates that

ranged from 0.0 0.2 d-1 following the addition of surplus nutrients, while Elser and

Frees (1995) reported changes in growth rates of 0.02-0.25 d-1 with the addition of

nutrients. The range in growth rates (0.25 0.53 d-1) and the range in chlorophyll-a

measured from the initial and final treatment subsamples (0.40-51.44 ug L1) in this study

are not unusual when compared to those reported for other systems. Growth rates (d-1)

reported in the literature surveyed ranged from 0.10 0.80 (Elser and Frees 1995, Hein

and Riemann 1995, Phlips et al. 2002), and Frazer et al. (2004) report chlorophyll-a

values that range from 0.2 74.4 ug L-1 in estuarine systems adjacent to three counties

along Florida's west coast.

Characterizing the two-dimensional response surfaces created in this study both

qualitatively and quantitatively, can provide valuable information in identifying ways of

managing nitrogen and phosphorus input to achieve management goals. For systems

where algal biomass is a primary contributor to water clarity, like Kings Bay, the

maximum biomass results of this study could be used to calculate the algal biomass that

could be expected given a particular nutrient reduction strategy. Currently, nutrient

concentrations in Kings Bay are relatively low in comparison to other coastal, spring-fed









systems in Florida (see Frazer et al. 2001). However, nutrient concentrations (nitrate in

particular) are elevated compared to the presumed historical values (see Jones and

Upchurch 1994). As point sources of nutrient load to the Kings Bay system have been

removed, additional nutrient reduction will require reduction of non-point sources. This

may prove to be a difficult task considering the rapid rate of population growth and the

associated changes in land-use within the broader Kings Bay watershed (Jones and

Upchurch 1994, SWFWMD 2003). This being the case, managers seeking to achieve an

increase in water clarity in Kings Bay may have to focus on other factors that influence

the accumulation of phytoplankton biomass such as removal processes.

In shallow estuarine systems like Kings Bay, phytoplankton are typically removed

by physical processes, e.g., flushing, and potentially by grazing (Phlips et al. 2002).

Although little is known about grazing effects in Kings Bay, there have been modeling

studies that characterize circulation patterns and calculate flushing rates. Open waters of

the bay are thought to have relatively short particle residence times, between 50-59 hrs (2

- 2.5 days), while dye injection studies suggest that Kings Bay is flushed every 71-94 hrs

(3 4 days), depending on the magnitude of spring discharge (Hammett et al. 1996).

Ambient phytoplankton growth rates measured in this study suggest that phytoplankton

can double in as little as 1.4 days. At present, phytoplankton doubling times are close to

the flushing rate. It may be possible to reduce phytoplankton growth rates to the point

where doubling times are greater than flushing rates, thereby potentially reducing the

accumulation of phytoplankton biomass within the bay. The magnitude of nutrient

limitation can be used to determine the reduction in nutrient concentration necessary to

achieve the desired response in phytoplankton growth rates. For example, the overall









magnitude of nitrogen limitation for site 81 estimates a 0.1011 change in growth rate per

mg L-1 of nitrogen. To increase doubling times from 1.4 days to 2 days, phytoplankton

growth rate would have to decrease from 0.4836 to 0.3466. The magnitude of nitrogen

limitation suggests that site 81 would have to undergo a 1.4-mg L-1 reduction of nitrogen

if flushing rates are the only factor considered to remove phytoplankton. However, the

nitrogen concentration in Kings Bay is already so low (TN -0.25 mg L-1, Hoyer et al.

1997), that a 1.4-mg L-1 reduction in nitrogen is not possible. A similar calculation with

phosphorus indicates that a decrease in phytoplankton growth rate from 0.4836 to 0.3466

at site 81 would require an estimated 0.12-mg L-1 reduction of phosphorus. The average

TP in Kings Bay is -0.029 mg L-1 (Hoyer et al. 1997), thus eliminating the reduction of

phosphorus as a possibility. Instead a more realistic approach to improving water clarity

in Kings Bay may include a combination of nutrient remediation strategies for reducing

phytoplankton as well as other means of improving water clarity such as sediment

stabilization or increased water flow.

An important factor to consider when using estimates like the ones calculated in

this study is the spatial and temporal variation of phytoplankton responses. When making

management decisions, it is not only important to understand what factors are affecting

the biogeochemical processes of the system, but where and how these processes are

affected. Although only two sites were used in this study, the results suggest that different

areas within the same system may be controlled by different limiting factors. This

phenomenon is not restricted to Kings Bay; Lake Okeechobee and Chesapeake Bay have

also been found to show similar spatial heterogeneity of nutrient limitation (Aldridge et

al. 1995, Fisher et al. 1999). It is important that these patterns, along with seasonal and






27


annual variations are taken into consideration in order to make effective management

decisions (Vanni and Temte 1990, Lewitus et al. 1998).















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BIOGRAPHICAL SKETCH

I am originally from St. Louis, MO. I moved to Florida to get my B.S. in marine

science at Eckerd College in St. Petersburg, FL. For my undergraduate thesis, I studied

the historic spatial and temporal changes in submerged aquatic vegetation in Tampa Bay,

FL, using aerial photography. After graduating, I spent a year working in the chemistry

lab at the Florida Wildlife Research Institute in St. Petersburg, FL. I then came to the

University of Florida to earn my M.S. under the advisement of Dr. Tom Frazer at the

Department of Fisheries and Aquatic Sciences.