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Nitrate-Nitrogen Dynamics in Tributaries of the Santa Fe River Watershed, North-Central Florida

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PAGE 1

1 NITRATE-NITROGEN DYNAMICS IN TRIB UTARIES OF THE SANTA FE RIVER WATERSHED, NORTH-CENTRAL FLORIDA By ADRIENNE E. FRISBEE 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 2007

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2 2007 Adrienne E Frisbee

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3 I would like to thankAun tieAdele Szablowski, who helped inst ill in me the belief that I could do this; my father, Joe Frisbee, whose unconditional love and support throu gh all of my journeys continues to amaze me; and my sister, Jeanine Fi rmin, my very best friend through it all.

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4 ACKNOWLEDGMENTS I would like to thank my advisor Mark Clark for his guidance and support, as well as my committee members, Richard Lowrance, Michelle Mack, and K.R.Reddy, and for their input on this project. I thank everyone in the Wetland Biogeochemis try laboratory, especially Yu Wang and Gavin Wilson for invaluable training with methods and instrumentation. I wish to thank Isabella Claret Torres for her input on sampling, methods, and statistics, as well as her support as a friend. I would like to thank Ed Dunne, Angelique Keppler, and Kani ka Inglett for their input as well. I would also like to thank Jenny Schafer fo r her immense help with editing. I would like to thank Jason Smith for many helpful biogeochemist ry talks and for help ing me get a job in California. I would like to thank the Univer sity of Florida Department of Animals Sciences and the Suwannee River Water Management District for financial support. Finally, I would like to thank my friends and family for their love and support. I am especially grateful to Hanna Lee, Melissa Lo tt and Jenny Schafer for their tremendous support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 BACKGROUND AND SI TE DESCRIPTION......................................................................12 Introduction................................................................................................................... ..........12 Nitrogen Removal in Riparian Wetlands................................................................................14 Regulators of Denitrification..................................................................................................15 Nitrogen and Floridas Waters................................................................................................18 The Santa Fe River Watershed...............................................................................................19 Objectives and Hypotheses.....................................................................................................20 2 WATER QUALITY MONITORING.....................................................................................25 Introduction................................................................................................................... ..........25 Objectives and Hypotheses..............................................................................................26 Materials and Methods.......................................................................................................... .26 Site Description...............................................................................................................26 Field Methods..................................................................................................................27 Analytical Methods.........................................................................................................28 Statistical Methods..........................................................................................................29 Results........................................................................................................................ .............29 Nitrate........................................................................................................................ ......29 Ammonium and Organic Nitrogen..................................................................................30 Dissolved Organic Carbon and Soluble Reactive Phosphorus........................................30 Chloride....................................................................................................................... ....31 Floodplain..................................................................................................................... ...31 Nitrate.......................................................................................................................31 Phosphate.................................................................................................................31 Discussion..................................................................................................................... ..........31 3 SOIL CHARACTERIZATION AND DENITRIFICATION.................................................52 Introduction................................................................................................................... ..........52 Objectives..................................................................................................................... ...52 Hypotheses..................................................................................................................... .53 Materials and Methods.......................................................................................................... .53

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6 Sampling Locations.........................................................................................................53 Soil Characterization Study.............................................................................................53 Field methods...........................................................................................................53 Laboratory methods..................................................................................................54 Denitrification potential...........................................................................................55 Nutrient Limitation Study................................................................................................56 Field methods...........................................................................................................56 Laboratory methods..................................................................................................56 Intact Core Study.............................................................................................................57 Field methods...........................................................................................................57 Laboratory methods..................................................................................................58 Statistics..................................................................................................................... ......58 Results........................................................................................................................ .............59 Soil Characterization Study.............................................................................................59 Nutrient Limitation Study................................................................................................61 Intact Core Study.............................................................................................................61 Discussion..................................................................................................................... ..........62 Soil Characterization Study.............................................................................................62 Nutrient Limitation Study................................................................................................63 Intact Core Study.............................................................................................................64 4 SUMMARY, IMPLICATIONS AND FUTURE RESEARCH.............................................76 Water Quality Monitoring......................................................................................................76 Soil Characterization an d Denitrification...............................................................................77 Future Research................................................................................................................ ......78 Conclusion..................................................................................................................... .........79 LIST OF REFERENCES............................................................................................................. ..80 BIOGRAPHICAL SKETCH.........................................................................................................85

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7 LIST OF TABLES Table page 2-2 Average percent change in nitrate per meter in tributary 1 (T1) for 11 months. Values with the same letter for significance level (SL) are not si gnificantly different.....44 2-3 Summary of NH4 + and TKN measured in tributary (T 1) and tributary 2 (T2) with one standard deviation in parentheses. Dashes indicate months that were not analyzed for NH4 + or TKN................................................................................................................47 3-1 Soil characteristics in tributary 1 (T1) and tributary 2 (T2) for the upland, bank, and stream. Values (n=15) represent me an and one standard deviation..............................68 3-2 Pearson product moment correlations (r value) between denitrification enzyme activity (DEA) rates and soil characteristics fo r tributary 1 and 2....................................70 3-3 Pearson product moment correlations (r value) between denitr ication rates of each treatment and soil characteristics.......................................................................................73 3-4 Mean one standard deviation of denitrif ication rates for each treatment in tributary 1 (T1) and tributary 2 (T2).................................................................................................73 3-5 Mean nitrate removal rate one standard deviation by sampling site. Mean values followed by the same value are not significan tly different. This analysis excludes a set of cores that were outliers.............................................................................................74 3-6 Pearson product moment correlations be tween nitrate removal rate per day and soil characteristics (DEA is denitrificati on enzyme activity, WEC and WEN are water extractable carbon and water extract able nitrogen, respectively)......................................74

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8 LIST OF FIGURES Figure page 1-1 Along the Ocala Uplift in eastern Florida, the Hawthorne layer becomes discontinuous, allowing surface and groundwater connections to occur...........................21 1-2 Location of the Santa Fe River Watershed in the Suwannee basin, also represented is the Cody Scarp (figure used with permission of J. Martin)...............................................22 1-3 Dominant land uses common in the Santa Fe River Watershed........................................23 1-4 A digital elevation map of the Santa Fe River Watershed that shows the repeating pattern of tributaries and where the river goes underground in the western portion of the watershed.................................................................................................................. ...24 2-1 Santa Fe River Beef Research Unit relativ e to Gainesville, Florida and the Santa Fe River watershed................................................................................................................ .36 2-2 The SFBRU cattle pastures with an orna mental plant nursery south of the property and tributaries that drain to the Santa Fe River floodplain................................................37 2-3 An example of Depositional Woody stream reach............................................................39 2-4 A Depositional Herbaceous reach......................................................................................39 2-5 An example of a Slightly Incised Woody reach................................................................39 2-6 Slightly Incised Herbaceous..............................................................................................40 2-7 Deeply Incised Woody.......................................................................................................40 2-8 An example of an Open Water reach.................................................................................40 2-9 A Moderately Incised Woody reach..................................................................................41 2-10 The Santa Fe River floodplain...........................................................................................41 2-11 Log of mean nitrate concentr ations of tributary 1 (T1) co mpared to tributary 2 (T2). Bars represent the standard deviation. T2 was not sampled in October due to the absence of surface water....................................................................................................42 2-12 Average and range of nitrate concentrati on in tributary 1 (T1) from headwaters to discharge for all months sampled......................................................................................43 2-13 Quantiles of monthly nitrate c oncentrations measured in T1............................................45

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9 2-14 Nitrate concentrations by season in tri butary 1 (T1). Spring = March, April, May; summer = June, August; fall = September, October, November; and winter = January, February.............................................................................................................. .46 2-15 SRP concentrations in tr ibutary 1 (T1), March 29, 2006...................................................48 2-16 SRP concentrations for tributary 2 (T2), March 29, 2006.................................................48 2-17 Chloride concentrations for May 20, 2005 along the length of tributary 1 (T1)...............49 2-18 Chloride concentrations for May 20, 2005 along the length of tributary (T2)..................49 2-19 Change in nitrate concentrations as tr ibutary 1 (T1) flows through the floodplain and improved pasture to the Santa Fe River.............................................................................50 2-20 Change in soluble reactive phosphorus (SRP ) concentrations from the last tributary 1 (T1) sample station, through the floodplai n and improved pasture to the Santa Fe River.......................................................................................................................... .........51 3-1 Sampling sites on each tribut ary. Each transect had samp le stations at the upland (U), bank (B), and stream channel (S)...............................................................................67 3-2 Mean denitrification enzyme activity (DEA ) rates in tributary 1 (T1) and tributary 2 (T2) in the upland, bank, and stream. Error bars represent one standard deviation..........69 3-3 Factor analysis of soil char acteristics and stream location................................................71 3-4 Mean redox potentials in tr ibutary 1 (T1) and tributar y 2 (T2). Nitrate is the dominant electron acceptor for redox potentials from 200-250mV. Error bars represent one standard deviation........................................................................................72 3-5 Mean + one standard deviation of denitrif ication rates for each treatment of a nutrient limitation experiment (N= nitrogen, N and C= nitrogen and carbon)...............................72 3-6 Mean nitrate removal rates + one standard deviation of each set of intact soil cores. The upland of the upper transect was not sa mpled because of equipment problems. Tubificid worms were found in the set of th ree cores taken from the channel of the middle transect................................................................................................................ ...73 3-7 Linear relationship between organic ma tter and denitrification enzyme activity (DEA) for the core soils.....................................................................................................75 3-8 Linear relationship between porosity and denitrification enzyme activity (DEA) rate for core soils................................................................................................................. ......75

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10 Abstract of Thesis Presented to the Graduate School of the Univ ersity of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NITRATE-NITROGEN DYNAMICS IN THE TR IBUTARIES OF THE SANTA FE RIVER WATERSHED, NORTH CENTRAL, FLORIDA By Adrienne E. Frisbee May 2007 Chair: Mark Clark Major: Soil and Water Science Nitrate runoff from agricultural systems is an increasing concern because of its potential effect on the health of both humans and ecosyst ems. Riparian systems have been shown to reduce nitrate concentrations in soil and water as a result of denitrifica tion processes that occur under anaerobic conditions. The Santa Fe River Basin in north central Florida contains many tributaries that drain adjacent agricultural system s and in the eastern part of the watershed that discharge to the Santa Fe or New Rivers. In ce ntral areas of the Santa Fe River, however, these tributaries on occasion discharge directly to the Floridian aquifer due to the karst and partially confined geology of the re gion. Increasing evidence suggest that nitrate concentrations in surface and groundwater are increasing, and in some instances have exceeded EPA safe drinking water standards. In an effort to better understand nitrate dynamics and denitr ification po tential of channel bed and riparian wetlands along tributaries of the Santa Fe River, a two year research investigation was established at Boston Farm-U F/IFAS Santa Fe River Beef Research Unit (SFRBRU). Fundamental questions addressed by this re search include 1) what are the seasonal dynamics of nitrate concentrations within two tributaries of the Santa Fe River, 2) are there differences in stream reach or stream fluvial morphology that influence nitrate assimilative

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11 capacity, 3) what effect does distance from stre am have on soil denitrific ation potential and 4) what effect does nitrate concentrat ion have on denitrificat ion potential within stream reaches. To answer these questions two stream s on the SFBRU were monitored. Results show little variation in nitrate c oncentration along a low nitrate concentration tributary. Along a high nitrate tr ibutary, however, concentrations were reduced an average of 31% from headwaters to discharge during the stu dy. Decreases in nitrate concentration were not uniform along the length of the stream, but instead indicate that several t ypes of stream reaches have significantly greater nitrate assimilative capacities than others. Soil characterization and denitrification studie s indicate that nitrate, carbon and anaerobic conditions are limiting denitrification in these tributaries.

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12 CHAPTER 1 BACKGROUND AND SI TE DESCRIPTION Introduction Nitrogen is the most abundant element on earth, ye t it is often the most limiting nutrient for plants and microbes in marine and terrestrial ecosystems (G oldman 1999, Casblanq 1999, Burns 1992). Nitrogen is limiting because it is predominantly present in the atmosphere as dinitrogen gas (N2), a nitrogen form unavailable to most organisms. Humans however, have dramatically increased the amount of available N on earth by a factor of 10 through anthropogenic and industrial N fixation (G alloway et al. 2004). In 1913, the Haber-Bosch process was developed to convert N2 to NH3 for fertilizer to improve food production. Combustion of fossil fu els along with the cultiv ation of rice, legumes, and other N-fixing crops has also increased biologically available forms of N, commonly in the form of ammonium (NH4 +) or nitrate (NO3 -) (Galloway et al. 2004). Inputs of available nitrogen dramatically increase plant productivity; however, with extens ive nitrogen loading to an ecosystem, more N may be available than plants and microbes can use (Aber et al. 1989). As excess nitrogen accumulates over time, it can have significant effects within an ecosystem. The Nitrogen Cascade refers to changes that occur as an ecosystem becomes saturated with nitrogen. There is an initial increase in productivity; how ever, over time, nitrogen loading has been shown to decrease biodiversity in forests, grasslands, lakes, and streams (Aber et al. 1995, Vitousek et al. 1997). Soil acidicification and a decrease in soil fertility may also occur because leaching of nitrate ions from the soil facilitates the rel ease of base cations su ch as calcium. There are a number of other de trimental effects that nitrogen accumulation can have on the growth and health of plants in natural and agroecosystems. Fo r instance, with an oversupply of nitrogen, excessive vegetative growth and plan t cell enlargement can cause a plant to become

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13 weak and top heavy. Other effects include dela yed plant maturity and reduced resistance to disease and pests (Bra dy et al. 2002). Excess nitrogen loading also has profound effect s on waterways. As streams, creeks, or rivers with elevated levels of dissolv ed organic nitrogen (DON), ammonium (NH4 +) or nitrate (NO3 -) drain into ponds, lakes, and oceans, eutrophi cation and degradation of water quality can occur (Seitzinger 1988). This can lead to algal blooms, fish kills change in species composition, and hypoxic conditions (Rabalais et al. 1996, van der Hoek 2004). Many zones of severe hypoxia occur where freshwater rivers high in nutrie nts enter coastal waters such as those near Louisiana, New York, New Jersey, Alabama, Texa s, and Florida leading to mass mortality of benthic communities and stress ed fisheries (Diaz 2001). Nitrate, an inorganic form of nitrogen, is uni que because it is a negatively charged ion, making it more susceptible to leaching than other positively charged nitrogen species that adhere to negatively charged soil particles. As nitr ate moves in water through the soil and enters ground and surface waters, it can have detrimental e ffects on humans, animals, and ecosystems. Concentrations of nitrate in dri nking water greater than 10 mg L-1 are considered a health hazard to humans and animals. Excess NO3 can cause methemoglobinemia or blue babys syndrome and has also been linked to brain tumors in children and to forms of stomach cancer (Forman 2004). Respiratory infections and problems relate d to thyroid metabolism are also effects associated with high nitrate le vels in drinking water (Follett and Follett 2001). Nitrate concentrations above 1 mg L-1 have also been shown to be toxic to amphibians and insects (Rouse et al. 1999).

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14 Nitrogen Removal in Riparian Wetlands Intact riparian ecosystems have been found to reduce nitrogen concen trations in surface and groundwater. The ability of these buffer areas to transform nutrients is important in streams adjacent to agriculture areas that drain to freshwater and marine systems subject to eutrophication (Lowrance 1992). A lthough these wetlands can be rela tively small in area, they can be a major zone for nitrog en retention in plants (Schaed e and Lewis 2006) or nitrogen transformation through denitrific ation (Fennessy and Cronk 1997). If ground or surface water comes in to contact with plant roots, riparian plants can take up nutrients from the water column or soil porewater thus providing a temporary sink for nitrogen. Schaede and Lewis (2006) found that increased N loading in a nitrogen limited system caused an increase in plant tissue %N and changes in the root to shoot ratio in plants due to increases in nutrient use efficiency and productivity. Yet, as plants senesce, most of the nitrogen will leach from the plant or be mineralized by microbes, rel easing it to the ecosystem. Plants can remove a significant amount of nitrogen from soil and water; thus, unless plants are harvested or a portion of the biomass accumulates as peat, they do not provide a long term sink for nitrogen. Denitrification, another process that removes n itrogen in riparian areas, takes place in soils and sediments under anaerobic conditions. This reaction occurs when facultative heterotrophic bacteria must use alternate electron acceptors during respir ation under low oxygen conditions. Nitrate is reduced to din itrogen, nitric and nitrous oxide gases that are lost to the atmosphere and thus nitrogen is removed from the water column. This is a long-term sink for nitrogen since th ese gases are only available to a few microorganisms during nitrogen fixation. As a result of flooded conditions, at least half of the denitrification on land has been found in wetland s, lake sediments, and riparian ecosystems (Bowden 1986).

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15 Restored wetlands in agricultural landscapes can be self-sustain ing and effective at removing excess N if properly managed. For the ma nagement of animal waste, denitrification in riparian areas can be a valuable process to remove N from liquid manure and other non-point source pollutants that are land a pplied (Lowrance et al. 1998). Cleaner water, however, may come at a cost to air quality. Denitrification plays a role in global climate change because it generates greenhouse gases. If NO3 is not reduced completely to N2, microbial by-products N2O and NOx will be the end products of denitrification. Production of N2O rather than N2 is favored at low pH (Johns et al. 2004), low temperatur e, and high oxygen and nitrate concentrations (Chapin et al. 2002). N2O has a long residence time in the atmosphere due to its low reactivity. This gas contributes to global warming since it ca n absorb infrared radiation and has the capacity to contribute about 300 times the greenhouse effect as one molecule of CO2 (Schlesinger 1997). Also, in reactions in the stratos phere, this produces NO, a gas that contributes to the destruction of good ozone. Another intermediate pro duct of denitrification is NOx. This is a very reactive gas that is involved in the production of st ratospheric ozone, or the photoch emical smog that is common in highly populated urban areas. Smog is known to cau se lung problems in humans. NOx is also a component of acid rain in the form of nitric acid. Not only is this a strong acid that decreases the pH of soils, it also deposits available N in ecosystems. Regulators of Denitrification Several factors influence where and at what ra te denitrification occu rs. Denitrification requires the presence of a labi le carbon source, anaerobic conditi ons, a nitrate source, and an active microbial community. Other abiotic fact ors such as temperature and soil texture can affect rates of denitrification.

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16 A number of studies have found the presence of a readily available carbon source to be the primary factor affecting rates of denitrification in ecosystems (Marienssen and Schops 1999). Under waterlogged conditions, the breakdown of orga nic matter is slow because, in the absence of O2, microbes must use an alternat e electron accept or such as CH4, NO3 -, Fe3+, Mn 4+, or SO4 2during respiration. These electron acceptors are not as energetically efficient as O2, which leads to a slower decomposition rate and the accumula tion of organic matter and, thus, electron donors for the denitrification process (DAngelo and Reddy 1999). Dissolved organic carbon (DOC), another source of carbon in riparian ecosystems, has been shown to be highly correlated with rates of denitrification (Desimone and Howes 1996). Moisture content also affects denitrification since anaerobic conditions must be present for denitrification to occur. Studies show that de nitrification rates have a significant relationship with moisture content (Schnabel et al. 1997, Schi pper et al. 1993). Schna bel et al. (1997) found that moisture content decreased with distance from streams in riparian areas and increased with soil depth; however, where moisture conditions ar e optimal, other factors such as carbon may be limited. Moisture content in soils is also affect ed by water table fluctuat ions, therefore seasonal or event driven changes in water table can str ongly influence the nitrogen cycle in the processes of nitrification, mineralization, a nd denitrification (Reddy et al. 1989 ). In areas subject to high loads of nitrogen, a fluctuating water table wi ll increase the nitrogen removal efficiency in riparian zones (Hefting et al. 2004). The process of denitrification is also controlled by the pr esence of a nitrate source. Systems that are flooded year-round must rely on the diffusion of nitrate from aerobic to anaerobic layers for denitrificati on to take place. If a system doe s not have a nitrate source, then denitrification may be controlled by nitrification rates. Nitrif ication is the process where NH4 + is

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17 oxidized to NO3 by autotrophic bacteria. This occurs in aerobic portions of the soil, and nitrate can diffuse to anaerobic soils along a concentration gradient. Even if microsites within the soil are anaerobic, seasonal water table fluctuations s timulate nitrification (Sch ipper et al. 1993). If O2 concentrations are too low and ni trification cannot occur, however, NO3 production can be a rate-limiting step of denitrification. Denitrification is indirectly affected by soil te xture. Higher rates of denitrification have been found in fine-textured soils rather than sandy so ils (Hefting et al. 2004). For instance, during storm events, flooded conditions ideal for deni trification can be shor t-lived because of the rapid drainage that occurs in coarse, sandy soil s. This is related to water filled pore space (WFPS); as WFPS increases so do rates of deni trification. Aulakh et al. (1992) found that denitrification only occurs at a WFPS of 60% and higher. All microbial processes are re gulated by temperature. Q10 is the rule of thumb that with every 10 degree increase in temperature, biologi cal activity will double. Denitrification is a mechanism carried out by microbes, and it has also been shown to be highly affected by temperature in lab studies (Fischer and Whalen 2005, Maag et al. 1997). The limiting factor for denitrification varies among ecosystems, as well as in microsites within an ecosystem. For instance, denitrification can take place in microsites of the soil profile if a soil is well-drained with s easonal wetting periods. This cr eates anaerobic hotspots within the soil profile where denitrification can take place. High carbon microsites are also hypothesized to be a major source of error for rate s of denitrification m easured. Knowledge of soil properties, hydrology, climate, and the biotic community can help predict how effective a system will be at removing nitrat e through denitrification. This ma y be especially important near agricultural areas with c onnections to groundwater.

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18 Nitrogen and Floridas Waters Waterways impacted with nitrates are especi ally problematic in areas where direct connections between ground and surface waters o ccur. These connections can lead to the contamination of aquifers, and, subsequently to sources of drinking wa ter, especially near industrial and agricu ltural landscapes. In many parts of Flor ida, connections can be numerous as a result of geology. Limestone from the states marine origins lies beneath Florida soils. A geological formation known as karst forms when limestone comes into contact w ith carbonic acid. When CO2 is dissolved in water from the break-dow n of organic matter, it forms carbonic acid (H2CO3). As carbonic acid comes into contact with limestone, calcium carbonate is easily dissolved. Over time, holes in the limestone deve lop from this erosion process forming karst. Some examples of karst formations in Florida are caves, springs, and sinkholes, all of which provide a conduit between su rface and ground waters. In most of Florida, this direct connectivity is not a concern because an impermeable layer of silt and clay, called the Hawthorne layer, und erlays the soil. The Hawthorne formation was formed by the deposition of phosphorus-rich clay and sand from ancient rivers and can be as deep as 800 ft in parts of western Florida. When the Hawthorne layer is in tact, there is no direct connection to the Floridan aquifer. In the nor thcentral portion of the state, however, along the Ocala Uplift, the Hawthorne layer has thinned so limestone is within 0 ft of the ground surface (Figure 1-1). The interface zone between intact and eroded Hawthorne layer is called the Cody Scarp. Along this interface, thinning of the Hawthorne layer allows increased infiltration of surface water to underlying limestone leading to dissolution and occasional collapse forming sinkholes. Once the Hawthorne layer is completely eroded, direct leaching of surface waters and rainfall through the soils to the aquifer is pos sible. Interaction between surface water and

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19 groundwater along this zone is sign ificantly increased and can lead to water quality degradation within the aquifer. Agriculture management practi ces in areas where karst formations are present can have significant ecological impacts on ecosystems and wa tersheds from applied fertilizers or animal waste. These non-point sources are subject to runoff and leaching into ground and surface waters, introducing nitrogen to waterways that otherwise might be nutrient limited. One area that illustrates the change in hydrologic connect ivity along the Cody Scarp and potential impacts of agricultural activities due to these conn ections is the Santa Fe River Watershed. The Santa Fe River Watershed The Santa Fe River watershed covers 3,585 square kilometers in north central Florida and drains into the 121 km long Santa Fe River. This watershed lies within the Suwannee River Basin that drains to the Gulf of Mexico (Figure 1-2). This area of Flor ida typically receives a mean annual precipitation of 1.3 meters and has a mean annual temperature of 24oC. Dominant land use types in th is area of Florida are silv iculture, row crop and pasture agriculture, and undeveloped natu ral areas (Figure 1-3). In the upper and middle watershed, agricultural and timber production ar eas are of concern because fertilizer and animal waste may be susceptible to runoff and leaching into tribut aries, creeks, and springs. Along the Santa Fe River, numerous tributaries drain agriculture areas that contribute water and nutrients to the river (Figure 1-4). Major tributaries include the Ichetu cknee, Olustee, New, and Sampson Rivers. Because of the geology of this region, these surface waters can come into contact with karst formations through sinkholes. The river actually enters a majo r sinkhole near the Cody Scarp and goes completely underground for 5 km and re-emerges before entering the Gulf of Mexico (Figure 1-4). Therefore surface waters in the upper and middle Santa Fe watershed will eventually enter groundwater and then eventually drain to fr eshwater and marine systems

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20 possibly leading to eutrophicati on, aquifer contamination, algal bl ooms, and other degradations in water quality. Possible nitrate sources in this watershed include se ptic tanks, atmospheric deposition, fertilizers, and animal waste. Objectives and Hypotheses The main goal of this study was to characteri ze water quality in several tributaries of the middle Santa Fe River watershed and to determ ine the extent to whic h riparian soils can effectively reduce nitrate concentrations in wate rs impacted by agriculture. Specific objectives were to evaluate spatial nitrat e-nitrogen dynamics in tributarie s and riparian wetlands at the Boston Farm Santa Fe River Ranch Beef Unit; identify reaches within the tributaries th at may have a greater capacity to remove nitrate; determine if carbon or nitrogen is limiting de nitrification in these riparian areas; determine the denitrification potential in a riparian wetland zone characteristic of tributaries in the Santa Fe River basin; Findings from studies that addressed thes e objectives are outlined in the following chapters of this thesis. In Chap ter 2, nitrate concentrations as we ll as other nutrients in surface waters of the two tributaries are discussed. Soil characteristics and denitrificat ion potential of soils along the tributary and adjacent wetlands are addressed in Chapter 3. Chapter 4 is a summa ry chapter to discuss implications of these finding and suggestions for future research.

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21 Figure 1-1. Along the Ocala Uplift in easte rn Florida, the Hawthorne layer becomes discontinuous, allowing surface and groundwater connections to occur. (Reprinted with permission from The Florida Speol ogical Society, Gainesville, Florida, http://www.caves.com/fss/pages/misc/geology.htm.)

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22 Figure 1-2. Location of the Sant a Fe River and Suwannee River, relative to the Cody Scarp (Reprinted with permission from Martin, J, Screaton, E., and Moore, P.2004. Surface and ground water mixing along the Cody Scarp: An example from the Santa Fe River Sink-Rise system. USGS Suwannee River Basin and Estuary Integrated Science Workshop Proceedings.

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23 Figure 1-3. Dominant land uses comm on in the Santa Fe River Watershed

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24 Figure 1-4. A digital elevation map of the Sant a Fe River Watershed that shows the repeating pattern of tributaries and where the river goes underground in the western portion of the watershed

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25 CHAPTER 2 WATER QUALITY MONITORING Introduction Numerous tributaries drain agri culture areas leading to the Santa Fe River that eventually drains to the Gulf of Mexico. High concentrations of nitrate in water can be detrimental to humans, and to marine or freshwater ecosystems. The research and development of methods to decrease nitrate in wate r is of interest especi ally where groundwater wi ll be impacted. If a sufficient riparian buffer exists along these tributar ies, there is the potential for nitrates and other nutrients to be reduced in the water column through denitrification or plant uptake. Water column nitrate concentrations may also decrease when freshwater systems are diluted by surface runoff or groundwater intrusion. Tributaries of the Santa Fe Ri ver may have spatial differences in water quality as a result of biotic and abiotic factors. For instance, some reaches of a tributary may have plants that are able to immobilize nitrate from the water column. Other areas may be anaerobic with a labile carbon source, conditions ideal for denitrificat ion. Other tributary reaches may be too channelized or sandy for significan t nitrogen removal to occur. As a resu lt, nitrate removal efficiencies in tributaries imp acted by agriculture ma y vary along the length of each tributary. Nitrate concentrations in tributaries draining agricultural areas may also have seasonal variations in water qualit y. For example, irrigation and ferti lization practices are maximized at different times of the year according to plant ne eds, which can cause nitrate concentrations to vary in tributaries. Precipitation and evapotranspiration can also alter tributary nitrat e concentrations by diluting or concentrating nitrates. While long-term studies are necessary to thor oughly understand seasonal changes, inferences can be made on what may be driving measured fluctuations over time.

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26 In order to better understand nitrate-nitrogen dyna mics in tributaries of the Santa Fe River Watershed, a one year study was conducted on two tri butaries that drain to the Santa Fe River at the Boston FarmSanta Fe Beef Research Unit (SFRBU). Objectives and Hypotheses The major goal of the water quality study was to understand the fate of nitrate in two tributaries that drain to the Santa Fe River. Specific objectives were to 1. make observations of how nitrate concen trations vary from month to month; 2. determine if different stream reaches remove more nitrate than others. Specific Hypotheses: 1. Nitrate concentrations will vary over the course of the study as a result of season changes, fertilization, or irrigation. It is e xpected that highest fe rtilization rates will be in spring and summer, and therefore hi ghest nitrate concentrations will occur during these months. 2. Some reaches in the tributaries will be mo re effective at removing nitrate from the water column than others. Reaches with pl ants in the water column or an available carbon source are hypothesized to remove more nitrate than sandy channel reaches without plants. Materials and Methods Site Description The Boston Farm Santa Fe River Ranch Beef Unit (SFBRU), a University of Florida property, provides an excellent representative site of typical landus e and topography along the middle third of the Santa Fe River. The resear ch site is located about 30 miles northeast of Gainesville, Florida in Alachua County (Figure 2-1). Soils in this watershed are sandy and are pre dominantly Ultisols, Spodosols, and Entisols. Specific soils in sampling areas are Sparr fine sand, Pelham, Plummer, and Masotte soils, and Chipley sand (SSURGO). The site has a number of features characteri stic of north central

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27 Floridas geologic and biotic communities. These include groundwater seeps, sinkholes, tributaries, ponds, and wetland communities. Land use on the site consists of low intensit y pastures bordered by forests and riparian areas associated with the tributaries. The rese arch unit supports a low density cattle operation with about 300 heifers on 1,600 acres. Adjacent to the property is a plant nursery that is potentially responsible for elevated nitrate conc entrations measured in water sampled from the site in 2004. Two tributaries run the length of the property and drain to a floodplain leading to the Santa Fe River (Figure 2-2). Tributary 1 (T1) drains cattle pastures on the Santa Fe Beef Research Unit SFBRU as well as The Holly Factory, an orname ntal plant nursery adjacent to the research site. During this study, cattle were only observe d in the pasture bordering this tributary during the month of October. Tributary 2 (T2) had less flow than T1 and at times went underground or had low water levels during the sampling period. Cattle are kept out of this tributary by barbwire fencing, although runoff can still enter the stream from nearby pasture and from upstream during larger rainfall events Field Methods To address the hypotheses posed in this study, two tributaries in the Santa Fe watershed were selected on the Santa Fe River Beef Re search Unit Boston Farm (Figure 2-2). Along these tributaries, we designated transitional zones between morphologically different stream reaches. Eight morphologically discrete str eam segments were designated using dominant vegetation type, degree of bank incision, and whet her depositional or erosional processes were the principal drivers along the reach (Table 2-1, Figures 2-2 to 2-9) These classifications can be compared to the widely used Rosgen stream classification system which uses shape, slope and

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28 pattern to classify streams and rivers (Rosge n and Silvey 1996). The Rosgen classification, however, does not take into account dominant vegetative community. Once stream reaches were classified accord ing to the above criteria, monthly water samples were taken at the beginning and end of each reach. This sampling method led to a total of 20 sampling stations along T1 and 10 along T2. For each sample station, an acid washed 250 mL bottle was rinsed three times with site water before collecting a sample. Care was taken to collect samples from an undisturbed portion of the water column in the middle of the channel at mid-water column depth. Water samples were th en acidified to a pH of 2 with ultra pure concentrated sulfuric acid and put on ice for tr ansport to the lab. In the lab, samples were transferred to scintillation vials. Samples to be analyzed for NO3 -, NH4 +, and DOC were filtered with a Whatman 0.45m filter. Samples analyzed for total Kjeldahl nitrogen (TKN) were not filtered. In May 2005 and March 2006, water samples were collected in T1 from the end of the tributary, through the floodplain and improved pasture up to the Santa Fe River, this resulted in an additional four samples. These samples were analyzed for NO3 -, SRP and Cl-. Analytical Methods All water samples were refrigerated and an alyzed for nutrients within 28 days as recommended by the EPA. Nitrate, ammonium and TKN were analyzed to determine the dominant nitrogen forms present in the water co lumn. Nitrate was analyzed colorimetrically using the cadmium reduction method on a rapid flow or a discrete analyzer (EPA method 353.2). Ammonium was analyzed colorimetrically on a Technicon AAIII autoanalyzer (EPA. 350.1). TKN was determined by digesting the water samp les with sulfuric aci d and a copper sulfate mixture to convert organic forms of nitrogen to ammonium. Ammonium was then analyzed colorimetrically on a Technicon AAII (EPA. 351.2).

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29 Dissolved organic carbon (DOC) and solubl e reactive phosphorus (SRP) were analyzed because of their potential roles in nutrient limita tion to plants and microbes, which can in turn affect the nitrogen cycle. DOC was analyzed on a Shimadzu TOC 5050a (EPA 415.1). SRP was measured colorimetrically on a spectrophotometer (EPA 365.1). Chloride concentrations were measured to determine if nitrate concentrations in the tributaries were being diluted by surface r unoff or groundwater intrusion. Chloride concentrations were analyzed on a Dionex Ion Chromatograph. All above methods used the QA/QC require ments set by the Wetland Biogeochemistry laboratory which require a spike, repeat, standa rd, and blank to be run for every 20 samples analyzed. Statistical Methods All statistics were analyzed w ith JMP IN 5.1. T-tests were used to compare two means, and ANOVAs followed by Tukey-Kramer tests were used when comparing more than one mean. All data were tested for a normal distribution and transformed if necessary before performing analyses. Results Nitrate For eleven months of sampling, nitrate conc entrations in T1 had an average nitrate concentration of 4.73 1.01 mg L-1 (mean SD). Nitrate concentrations in T2 were significantly lower than nitrate co ncentrations in T1 (p< 0.001) fo r all months, with an average of 0.03 0.03 mg L-1 (Figure 2-10). Nitrate concentrations in T2 were consiste ntly low and showed no significant spatial or temporal variability. Therefore, the remainde r of this chapter will focus on T1 for further

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30 analyses of tributary nitrat e-nitrogen dynamics. Nitrate c oncentration decreased by 13% from headwaters to discharge in T1 with an average reduction of 31% (Figure 2-11). The Open Water (OW), Depositional Herbaceous (DH), Moderately Incised Herbaceous (MIH), and Slightly Incised Woody (SIW) reaches all removed significantly more nitrate per meter than the other six reach designations (Table 2-2). Because of the relatively short sampling period, statistics were not performed to determine if differences existed between months or s easons. There were, however, considerable differences in mean tributary nitrate concentr ation between months and seasons that may be related to fertilization, irriga tion, cattle grazing, or seasonal c limate differences (Figures 2-12 and 2-13). Ammonium and Organic Nitrogen TKN and NH4 + were not significantly different in the two tributaries (Table 2-3). T1 had NH4 + concentrations of 0.14 0.11 mg L-1 and TKN concentrations of 0.51 0.32 mg L-1. T2 had NH4 + concentrations of 0.22 0.23 mg L-1 and TKN concentrations of 0.43 0.29 mg L-1. Dissolved Organic Carbon and Soluble Reactive Phosphorus T2 DOC concentrations were significantly hi gher than T1 concentr ations (p= 0.005). T1 had an average DOC concentration of 5.79 mg L-1 1.59, whereas T2 had an average of 12.94 mg L-1 8.69. SRP concentrations were analyzed for the Ma rch 2006 sample event. SRP concentrations were not significantly different in the two tr ibutaries (p= 0.5). In T1, SRP concentrations decreased 72% along the length of the tributary (Figure 2-14). In T2, SRP was reduced by 60%, but increased when the tributary reac hed the floodplain (Figure 2-15).

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31 Chloride Chloride concentrations measured in T1 re mained similar along the tributary until the sampling station just before the floodplai n where it increases from 6.5 to 13.0 mg L-1 (Figure 216). Chloride concentrations were more variab le in T2, with a maximum concentration of 8.2 mg L-1 (Figure 2-17). Floodplain Nitrate Nitrate in T1 was reduced another 81% in May 20, 2005 and 86% in March 29, 2006 from the last station in T1 through the floodplain and improved pasture to the Santa Fe River (Figure 2-18). Phosphate March 29, 2006, SRP was reduced 10% as T1 went through the floodplain to the river (Figure 2-19). Discussion T1 and T2 were not significantly different in NH4 + or TKN concentrations. NO3 concentrations, however, were significantly higher in T1 than in T2. Because both tributaries are bordered by cattle pastures, T1 is believed to be significantly higher in nitrate as a result of runoff from landuse practices in th e upper watershed which in Figure 2-2 can be identified as a horticultural nursery. T1 receives irrigation and storm water from th e nursery, which is fertilized year round with NH4NO3, urea, and KNO3 (T. Stevens personal communication 2006). Nolan and Stone (2000) sampled over 50 sites across the United States and found that the major source of nitrogen to groundwater was found to be from fertilizers rather than manure or atmospheric deposition. Average nitrate conc entrations in groundwater were shown to be highest near agriculture areas (3.4 mg L-1) when compared to urban areas (1.6 mg L-1) and major aquifers

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32 (0.48mg L-1) (Nolan and Stone 2000). Although our study did not address nutrients in groundwater, it did find T1 surface wate rs to be impacted certain types of agricultural runoff. Over a year of monitoring, T1 consistently showed a reduc tion of nitrate in the water column as water moved from headwaters to discha rge. Many studies have shown that riparian areas can reduce nitrates in runoff before it reaches freshwater systems (Fennessy and Cronk 1997). This study, however, shows a reduction in nitrat es in the stream channel. This may be a result of plant uptake, denitrification, or dilution by ground or surface waters. Chloride concentrations measured in this tributary showed no major change along the length of T1 suggesting that the decrease in nitrate concentra tions is not due to a dilution by groundwater or surface runoff, but rather from plan t uptake or denitrification. Studies have shown phytoplankton and plants remove substan tial amounts of nitrogen from water systems (Schaede and Lewis 2006, Bledsoe et al. 2004). Phlips et al. (2002) found phytoplankton in the Indian River Lagoon, Florid a were most often limited by nitrogen. Phytoplankton populations were frequently observe d at the SFBRU in the Open Water reach and in the floodplain (in winter), thus phytoplan kton may provide a sink for nitrate in T1. Phytoplankton has also been shown to increase ra tes of denitrification by providing a labile carbon source to microbes (Sirivedhin and Gray 2006) Reaches with a closed tree canopy, however, are likely light limited, which would in hibit phytoplankton growth. Indeed, reaches with woody species showed little to no n itrate removal in th e water column. Smialek et al. (2006) showed higher rates of denitrification in soils with herbaceous species ( Juncus sp ) when compared to soils with woody species ( Salix sp ) present. Both woody and herbaceous plant species occu r along the tributaries, and aquati c plant species grow in some

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33 stream reaches. Both plant and algal species in th ese tributaries may have a role in the decrease in nitrate concentrations of the water column. The decrease in nitrate was not uniform along the length of the tributary; some reaches removed more nitrate than others, while some re aches released nitrate into the water column. The Open Water, Depositional Herbaceous, and Moderately Incised Herbaceous reaches removed the most nitrate from T1 during the year of monitoring. Numerous characteristics may contribute to high nitrate removal in the Open Water. For inst ance, water has a long residence time in this reach, which increases contact time with soils, plants and phytoplankton. The long residence time also leads to deposition and build up of organic matter. The sediments in the stream channel are constantly flooded and likel y anaerobic, making this an ideal location for denitrification. Finally, this reach has a number of species of aquatic pl ants along the edges of the tributary that may be assimilating nitrate. The Depositional Herbaceous reach is a portion of the tributar y that braids through organic and mineral deposits. These depositional areas ha ve built up over time, and a number of plant species are present. As water braids through these zones, it ma y come into contact with plant roots that take up nitrate. The plants can also provide a carbon source for denitrification. The Moderately Incised Herbaceous reaches have riparian and aquatic plants present that can take up nitrate. These reaches may also be receiving DOC as it leaches from the upland. It was, however, unexpected that this reach woul d remove a significant quantity of nitrate. Nitrate concentrations in T1 di d appear to vary over the cour se of the year. Highest mean concentrations were observed in October 2005, th e only month that cattle were observed in the pasture directly adjacent to the tributary. As a result, nitrate concentrat ions did not decrease much along the length of the tributary. Spring was found to have the hi ghest initial nitrate

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34 concentrations in the water column. This likely corresponds with the higher fertilizer application rates at the beginning of the grow ing season at the nursery upstream (T. Stevens, nursery owner, personal communication 2006). The most nitrat e removed along the length of T1, however, occurred in April. This is likely the result of warming soil temperatures and plant growth, which occur in the spring. On the other hand, the le ast amount of nitrate was removed along the length of the T1 in the fall. This may correspond with the release of nitrogen that occurs as plants senesce at the end of the growing season. Carbon quality and quantity is important to nitrogen cycling in aquatic environments because of its effects on denitrification, nitrif ication, and mineralization. Strauss and Lamberti (2000) found that glucose and leaf leachates inhib ited nitrification because heterotrophic bacteria outcompeted the chemoautotrophic bacteria respons ible for nitrification. DOC was found to be significantly higher in T2 than T 1, and this may inhibit nitrificat ion in this tributary. On the other hand, DOC may be providing a carbon source for denitrification in T1. Finally, if a stream has high carbon and low nitrogen, most inputs of n itrogen will be rapidly assimilated into plant and microbial biomass (Schlesing er 1997). All of these processe s could explain in part why nitrate concentrations were significantly lower in T2 than in T1. Available phosphorus concentratio ns in the water column in T1 decreased from headwaters to discharge. Microbial and plant uptake may both have a pa rt in SRP removal from T1. Phosphorus may also be adsorbing on to the surface of stream sediments. Nitrate and SRP concentrations were bot h dramatically reduced in May 20, 2005 and March 29, 2006 in the floodplain. This may be due to a combination of denitrification, dilution, and plant uptake. Although it is unknown what process is reduci ng nutrient concen trations, it is

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35 clear that the floodplain is impor tant in removing nitrate and SR P from agriculture impacted waters in T1. Overall, unlike T2, T1 waters were impacted by agricultural runoff. Nitrate concentrations, however, were reduced as T1 moved from headwaters to discharge in the floodplain. No change in chloride concentratio n along T1 suggests this reduction in nitrate is from denitrification or plant uptake rather than dilution from groundwater intrusion. SRP concentrations were also reduced al ong the length of the tributary. Some nitrate reaches were more effective at nitrate removal than others, likely due to differences in carbon availabili ty, retention time, and plant community. Nitrate was also significantly reduced as T1 passed through the floodplain.

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36 Figure 2-1. Santa Fe River Beef Research Unit relative to Gainesville, Florida and the Santa Fe River watershed. Gainesville Study Site

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37 The Holly Factory The Holly Factory UF / Boston Farm Santa Fe Beef Unit UF / Boston Farm Santa Fe Beef Unit Santa Fe River Santa Fe River CR 241 CR 241 Figure 2-2. The SFBRU cattle pastures with an ornamental plant nursery south of the property and tributaries that drain to the Santa Fe River floodplain. Tributary 1 Tributary 2

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38Table 2-1. Summary of stream reach characteristics. Reach typeAcronymDominant vegetation Depositional or Erosional Degree of Bank Incision Rosgen equivalentFigure Depositional woody DW tree species specificly Carya sp., Pinus sp., Quercus sp., Magnolia grandiflora depositionalnoneD or DA2-2 Depositional herbaceous DH herbaceous plant species such as Saururus cernuus, Juncus sp., Cephalanthus occidentalis, Hydrocotle umbellata, and Polygonum sp. depositionalnoneD or DA2-3 Slightly incised woody SIW tree species specificly Carya sp., Pinus sp., Quercus sp., Magnolia grandiflora erosional<30cmB2-4 Slightly incised herbaceous SIH herbaceous plant species such as Saururus cernuus, Juncus sp., Cephalanthus occidentalis, Hydrocotle umbellata, and Polygonum sp. erosional<30cmB2-5 Moderately incised woody MIW tree species specificly Carya sp., Pinus sp., Quercus sp., Magnolia grandiflora erosional<50cmA2-6 Deeply incised woody DIW tree species specificly Carya sp., Pinus sp., Quercus sp., Magnolia grandiflora erosional>50cmAa+2-7 Open waterOWaquatic emergent and floating plantsdepositionalnoneF2-8 FloodplainFP tree species such as Taxonium distichum and Nyssa sylvatica depositionalnoneF2-9

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39 Figure 2-3. An example of Depositional Woody stream reach. Figure 2-4. A Depositional Herbaceous reach. Figure 2-5. An example of a Slightly Incised Woody reach.

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40 Figure 2-6. Slightly Incised Herbaceous Figure 2-7. Deeply Incised Woody. Figure 2-8. An example of an Open Water reach

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41 Figure 2-9. A Moderately Incised Woody reach. Figure 2-10. The Sant a Fe River floodplain.

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42 Month M a r 0 5 A p r 0 5 M a y 0 5 J u n 0 5 A u g 0 5 S e p 0 5 O c t 0 5 N o v 0 5 J a n 0 6 F e b 0 6 M a r 0 6 Nitrate mg L-1 0.001 0.01 0.1 1 10 T1 T2 Figure 2-11. Log of mean nitrate concentrations of tributary 1 (T1) compared to tributary 2 (T2). Bars represent the standard deviation. T2 was not sampled in October due to the absence of surface water.

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43 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 02004006008001000 Distance (m)NO3mg L -1 Figure 2-12. Average and range of nitrate con centration in tributary 1 (T1) from headwaters to discharge for all months sampled Samples were collected at transitional poi nt between classified stream reaches. The reach classifications are as follows: DW= Depositional Woody, SIW= Slightly Incised Woody, MIW= Moderately Incised Woody, MIH= Moderately Incised Herbaceous, SIH= Slightly Incised Herbaceous, DH= Depositiona l Herbaceous, OW= Open Water, DIH= Deeply Incised Herbaceous, and FP= Floodplain. SIW DH OW FP MIW DH SIH MIH MIW MIW DIW DW SIW MIW MIW SIW DIH

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44 Table 2-2. Average percent change in nitrate per meter in tributary 1 (T1) for 11 months. Values with the same letter for significance level (SL) are not si gnificantly different. Reach TypeMeanSDSLOpen water0.270.21a Depositional Herbaceous0.130.65ab Moderately incised herbaceous0.080.09abc Slightly incised herbaceous0.040.10abc Deeply incised herbaceous0.010.02c Floodplain0.000.14c Moderately incised woody-0.010.36bc Deeply incised woody-0.040.20bc Slightly incised herbaceous-0.040.42abc% m-1

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45 Month M a r 0 5 A p r 0 5 M a y 0 5 J u n 0 5 A u g 0 5 S e p 0 5 O c t 0 5 N o v 0 5 J a n 0 6 F e b 0 6 M a r 0 6 NO 3mg L-1 0 2 4 6 8 Figure 2-13. Quantiles of monthly nitrate concentrations measured in T1.

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46 2 3 3 4 4 5 5 6 6 7 02004006008001000 Distance from headwaters (m)Nitrate (mg L-1 ) spring summer fall winter Figure 2-14. Nitrate concentratio ns by season in tributary 1 (T1) Spring = March, April, May; summer = June, August; fall = September, Oc tober, November; and winter = January, February.

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47 Table 2-3. Summary of NH4 + and TKN measured in tributary (T 1) and tributary 2 (T2) with one standard deviation in parentheses. Dashes indicate months that were not analyzed for NH4 + or TKN. Month Mean [NH4+]Mean [TKN] Mar-050.15 (0.08)0.32 (0.07) Apr-050.22 (0.08) May-050.22 (0.24) Jun-050.20 (0.35)0.50 (0.32) Aug-050.26 (0.31) Feb-06 0.86 (0.25) Mar-060.06 (0.02)0.26 (0.16) Average0.14 (0.11)0.51 (0.32) Mar-050.25 (0.11)0.41 (0.16) Apr-050.13 (0.03) May-050.27 (0.04) Jun-050.09 (0.20)0.30 (0.05) Aug-050.05 (0.03) Feb-06 0.63 (0.35) Mar-060.04 (0.01)0.22 (0.04) Average0.22 (0.23)0.43 (0.29) mg L-1T2 T1

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48 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 01002003004005006007008009001000 Distance along tributary (m)SRP mg L-1 Figure 2-15. SRP concentrations in tributary 1 (T1), March 29, 2006. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 050100150200250300350400450 Distance along tributary (m)SRP mg L-1 Figure 2-16. SRP concentrations for tributary 2 (T2), March 29, 2006.

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49 0 2 4 6 8 10 12 14 02004006008001000 Distance along tributary (m)Cl(mg L-1) Figure 2-17. Chloride concentrations for Ma y 20, 2005 along the length of tributary 1 (T1). 0 1 2 3 4 5 6 7 8 9 10 050100150200250300350400450 Distance along tributary (m)Cl(mg L-1) Figure 2-18. Chloride concentrations for Ma y 20, 2005 along the length of tributary (T2).

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50 0 1 2 3 4 5 6 020040060080010001200140016001800 Diatance (m)[Nitrate] mg L-1 May-05 Mar-06 Figure 2-19. Change in nitrat e concentrations as tributary 1 (T1) flows through the floodplain and improved pasture to the Santa Fe River. Distance (m) T1 before flood p lain After Flood p lain Following improved p asture Santa Fe River After more improved pasture and floodplain

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51 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 020040060080010001200140016001800 Distance (m)SRP mg L-1 Figure 2-20. Change in soluble reactive phosphorus (SRP) concentra tions from the last tributary 1 (T1) sample station, through the floodplai n and improved pasture to the Santa Fe River. T1 before flood p lain After Flood p lain Following improved p asture After more improved pasture and hardwood forest Santa Fe River

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52 CHAPTER 3 SOIL CHARACTERIZATION AND DENITRIFICATION Introduction Denitrification, a major removal pathway fo r nitrogen from ecosystems, requires low oxygen, a labile carbon source, a nitrate source, and an active microbial community. Because wetlands are often anaerobic with an accumulation of carbon, these ecosystems can provide ideal conditions for denitrification. Nitrate-nitrogen concentrations in a tribut ary impacted by agricultural runoff are being reduced by some mechanism as water moves along th e length of the tribut ary (Chapter 2). To determine the possible role of denitrification in reducing nitr ate in this system, three studies were conducted on two tributaries at the Santa Fe Beef Research Unit (SFBRU). A soil characterization study, a nutrient li mitation study, and an intact co re study were conducted on soils of the stream, bank, and upland of tribut ary 1 (T1) and tributary 2 (T2). These studies were conducted to investigate so il characteristics that have been shown to directly or indirectly influence denitrification in soils such as carbon and nitrogen (Fischer and Whalen 2005, Lowrance 1992, Aber et al. 1991). Objectives Specific objectives were to 1. determine if denitrification is a major rem oval pathway for nitrogen from the soils of the SFBRU; 2. determine what nutrients, if any, are li miting denitrification in this system; 3. determine if there are differences in nitrate removal rates among upland, bank, and stream channel soils in intact cores; 4. determine if nitrate removal rates in int act soil cores correspo nd to denitrification rates measured by the soil slurry method.

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53 Hypotheses Specific hyptheses were that 1. denitrification is a dominant pathway fo r nitrogen loss under anaerobic conditions; 2. because soils are mostly sandy in this region of Florida, carbon limits denitrification; 3. the bank and upland will have higher rates of nitrate removal from the water column than the channel due to mo re available soil carbon; 4. denitrification measurements using intact so il cores will be more variable than the soil slurry method because the experime nt is conducted in a less controlled environment. Materials and Methods Sampling Locations Soil sampling was conducted along three transect s established on a high nitrate (T1) and a low nitrate (T2) tributary at the SFBRU. All tran sects run perpendicular to the tributary and are located at the upper (headwater), middle, and lo wer portion (near floodplain) of each tributary. Five sampling stations along each tr ansect were established at the center of the stream channel, on either bank of the main channel and 25 meters up land from each bank sampling point (Figure 3-1). Bank sampling stations were repres entative of riparian areas, a nd the site 25 meters from the bank was representative of upland areas. These samp le sites and transects were used for all soil sampling events. Soil Characterization Study Field methods On June 20, 2005, triplicate soil samples were ra ndomly taken within a 1 meter radius of each sampling location along each transect. A 7 cm diameter soil corer with sharpened metal head was used to extract a soil sample to a depth of 5 cm, making sure to minimize compaction of the soil. A knife was used to cut any roots and to en sure the sample obtained was flush with the end of

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54 the soil corer. Each sample was extruded into a plastic storage bag and excess air was removed before sealing the bag. A total of 90 samp les were put on ice for transport to the lab. In addition to soil sampling, redox potential of the soil was measured four times between November 2005 and March 2006. Redox potential is a measure of how reduced a soil is and therefore what dominant electron acceptor is being used by microbes du ring respiration. To measure redox, platinum electrodes were set up in duplicate at sample sites along the middle transect of each tributary. Each electrode was in serted to a depth of 5 cm the depth at which all soil cores were taken. An Accumeter redox probe was connected to a pH meter and a platinum electrode to measure soil redox pot ential. All values were adju sted to the standard hydrogen electrode by adding 207mV to the measured value. Laboratory methods Once in the lab, soils were weighed for bul k density. Soils were then processed by homogenizing samples and removing any large live and dead plant material, roots, or rocks. Samples were stored in sealed plastic tubs at 4oC until analysis. Soil moisture content was measured grav imetrically by drying 10g of soil at 70oC for at least 72 hours. Samples were then reweighe d and soil moisture content was calculated. Soil organic matter content was determined by the loss on ignition method. Oven dried soil samples were ground and passed through a #60 sieve (0.25mm). Any soil remaining in the sieve was ground with a mortar and pestle until it could pass through the sieve. Approximately 2g of dry soil was placed in an aluminum tin, wei ghed, and combusted in a muffle furnace for 30 minutes at 250oC then for 3 hours at 550oC. Ashed samples were reweighed to determine total loss of organic matter (Jackson 1993).

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55 Soil pH was measured by placing 10 grams of soil and 10 grams of deionized water in a beaker and allowing the mixture to equilibrate fo r 30 minutes. The soil-water solution was then measured with a calibrated pH meter (Thomas 1996 and Hanlon 1984). To measure water extractable carbon and nitrate 2.5g of soil and 25mL of distilled deionized (DDI) water were added to plastic extr action tubes. A rubber st opper was placed in each tube and the soils were shaken on an end to end shaker at an intermediate speed for one hour. Samples were then centrifuged for 10 minutes at 6000 rpm. Extractions were filtered with a Whatman # 41 filter (0.45m). Water extractable nitrate (WEN) was analyzed by the cadmium reduction method discussed in chapter 2. Wate r extractable carbon (WEC) was measured on a Shimadzu TOC 5050A. Denitrification potential To measure denitrification potential of thes e soils, denitrification enzyme activity (DEA) was measured within 2 weeks of soil collection. This process meas ures the activity of microbes, specifically denitrifying bacteria under anaerobic conditions. For the DEA procedure, 8 g of soil was weighed into a 120mL glass serum bottle. Bottles were capped, crimped, and evacuated with N2 gas to establish anaerobic conditions. Five milliliters of purged H2O were added to create a soil slurry. The acetylene block method was used because acetylene gas (C2H2) blocks the final step in denitrification when N2O is reduced to N2. Acetylene was generated by adding water to calcium carbide rocks which immediately produces high gr ade acetylene gas. Twenty milliliters of acetylene gas were injected into each serum bottle Samples were put on a shaker for 30 minutes to ensure complete mixing of acetylene throughout the soil. Eight milliliters of DEA solution (288 mg L-1 glucose, 56 mg L-1 KNO3, and 100mg L-1 chloroamphenicol) were added to each sample and soils were put on an end to end shaker to inc ubate in the dark at a constant temperature of

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56 25oC. The volume of chloroamphenicol used in this study was selected based on experiments conducted by Murray and Knowles (1999). Gas sa mples were collected every 30 minutes for organic soils and every hour for sandy soils for up to 3 hours. Samples were stored in 4 milliliters evacuated, crimp-top, glass, serum bottles until analysis. N2O gas samples were measured on a Shimadzu gas chromatograph 14A with a 63Ni electron capture detector Column temperature was 30oC, detector temperature was 240oC, and injector temperature was 120oC. The carrier gas was Argon and 5% methane. Denitrification rates we re obtained by calculating the slope of the line obtained when gas concentrations were plotted over time. Nutrient Limitation Study Field methods Using the same soil transects discussed a bove, in January 2006, tr iplicate soil samples were taken to a depth of 5 cm in the stream cha nnel, at the east bank, and at the east upland soil sampling locations. Triplicate samples at each location were combined into one sample. Samples were homogenized and stored in a sealed plastic bag on ice for transport to the lab. Laboratory methods Soil samples were processed and moisture content and loss on ignition determined according to the methods described above. To determine what may be limiting denitrification rates in this system, 8-10g of soil from each samp le were added to 3 serum bottles to represent each treatment: ambient, + nitrogen (+N), and + nitrogen + carbon (+N+C). Each serum bottle was capped, crimped, and flushed with N2. Five milliliters of N2 purged water and 20 milliliters of acetylene were added to each sample as described above. For ambient samples, 8 milliliters of DDI water were added to the serum bottles, and samples were set to incubate in the dark on an end to end shaker at 25oC. Based on previous sampling, the ambient soils presumably had low nitr ate concentrations, so nitrate consumption was

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57 expected to occur quickly. Gas samples were ta ken at approximately 20 min, 40 min, 2 hours, and 4 hours. For the +N treatment, 8mL of a 5mg L-1 nitrate solution were added to each serum bottle. This concentration was chosen because it is simi lar to the average nitrat e concentration of the water sampled in T1. Samples were set to incuba te in the dark on an end to end shaker at 25oC. Gas samples were taken at 1, 2, 4, 16 and 48 hours. These sample times were selected to try to catch the linear portion of the denitrification reaction. For the +N+C treatment, 8 mL of 5 mg L-1 nitrate solution and 4 grams of ground litter as a carbon source were added to each sample. Litter wa s collected from the sample site near the stream channel and was composed of a mix of woody (pine and oak) and herbaceous (knotweed, Juncus sp and grass) species. Samples were in cubated in the dark on a shaker at 25oC. Based on the analysis of the +N gas samples, samples for the +N+C reaction were sampled at: 1.5, 3, 10, 13, and 28 hours. All gas samples were stored in evac uated 4mL glass serum bottles. The N2O gas concentration of each sample was measured on a Shimadzu 14A gas chromatograph. Intact Core Study Field methods To measure nitrate removal capacity of SFBRU soils, an intact core study was carried out in April 2006. Triplicate intact soil samples were taken along three transects in the T1 tributary (described in Chapter 3) within the stream ch annel, east bank, and east upland of the impacted tributary. Soil cores were also taken in the flood plain to determine the nitrate removal rate of floodplain soils. Each soil core was taken to a depth of 5 cm with a sharpened steel head placed on a 35 cm long, clear polycarbonate tube. Care was taken to minimize compaction when the

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58 apparatus was either pushed or hammered into the soil. The steel head was removed and both ends of the tube capped for transport. All collected co res were transported upright, on ice to the lab. Laboratory methods Site water from T1 with an initia l nitrate concentration of 6.21 mg L-1 was added until each soil core was saturated and covere d with 20cm of water. All fl ooded cores were placed in an aquarium filled with water to moderate ambien t temperature changes and maintain a neutral hydraulic head difference between th e inside and the outside of the core The water column of each core was mixed by continuous bubbling with ambient air pumped through tubes fixed with a 1.5 gauge hypodermic needle. Bubbling rate was su fficient to keep the wa ter column mixed and under aerobic conditions, but not to the level that sediments became suspended. Black plastic was placed over the entire experiment to minimize light a nd, therefore, deter algal gr owth in the cores. Water samples and temperature readings were collected from the water column 14 times over 8 days (time sampled = 0, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, and 192 hours). Samples were analyzed with the Cadmiu m reduction method on an AQ2, a discrete autoanalyzer, to measure nitrate concentr ations in the water over time. Following completion of the experiment, soils from the intact cores were analyzed for organic matter, moisture content, and deni trification enzyme activity rate (DEA). Statistics All statistical analyses were performed in JMP IN 5.1, Sigma Plot 8.0, or Statistica. To test differences between tributaries, a t-test wa s performed. To compare differences between denitrification rates by stream location, when comparing more than two means, ANOVAs followed by a TukeyKramer test were used. ANOVAs and TukeyKramer tests were also used to compare differences between treatmen ts in the nutrient limitation study.

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59 DEA rates were correlated with soil characterist ics to see what factors if any had an affect on denitrification. Correlations were also analyzed for soil properties and denitrificati on rates for the treatments. All Pearson product moment corre lations were performed in JMP IN 5.1 and Statistica. A factor analysis was performed by the Prin ciple Component extrac tion method to get an overview of how soil characteristics affect variati on in soils samples by location. Factor analyses were run with Statistica. Differences between soil cores were analy zed with ANOVAs followed by a TukeyKramer test in JMP 5.1. Correlati ons were run in JMP 5.1. Results Soil Characterization Study Soil bulk density, pH, % moisture content, % organic matter, water extractable carbon (WEC), and water extractable nitrate (WEN) measurements were used for initial soil characterization (Table 3-1). When combini ng sites along each trib utary, bulk density was significantly higher in T1 than in T2 (p= 0.04). WEN and WEC we re not significantly different (p= 0.13 and 0.10, respectively). Finally, % organic ma tter, % moisture content, and pH were not significantly different for T1 compared to T2 (p= 0.22, 0.57, and 0.12, respectively). Each tributary had a number of difference s in soil properties between upland, bank and stream soils. Differences between locations were observed for all soil properties except pH in T1 and moisture in T2 (Table 3-1). Overall, the mean DEA rate was 5.89 9.83 mg N2O kg soil-1 d-1. T1 had an average DEA rate of 8.73 12.78 mg N2O kg soil-1 d-1which was significantly higher than T2, with an average DEA rate of 2.50 2.68 mg N2O kg soil-1 d-1 (p= 0.04).

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60 For both tributaries, the upland and bank soils had significantly higher DEA rates than the stream channel soils (p<0.001; Figure 3-2). There were no differences in DEA rates between transects on either tributary. Percent organic matter had the strongest correlation with T1 and T2 DEA rates. DEA rate was also correlated with bulk density and WEC in both tributar ies. There was not a strong relationship in either tributary between DEA rate and pH, WEN, or % moisture content (Table 3-2). A factor analysis was performed by the Pr inciple component extraction method to get an overview of how these soil characteristics affected variation in soils samples by location (Figure 3-3). Factor 1 describes 49% of the variability in soil properties, and the parameters selected were % organic matter, DOC, DEA rate, and bulk dens ity. Percent organic matter, DOC, and DEA varied together, whereas bulk dens ity was inversely related to th ese soil properties. Factor 2 describes 24% of the variability in soil characteri stics, and the parameters selected by the Factor analysis were moisture content and soil NO3 -. These parameters were inversely related. Upland soils were most strongly influe nced by organic matter and soil nitrate concentration. Bank soils overlapped with all soil ch aracteristics, but clusters existed near soil nitrate and moisture content. Finally, stream soils were inversely rela ted to DOC, and organic matter, but positively related to bulk density (Figure 3-3). Redox potentials were not significantly different between T1 and T2 (p= 0.21, n= 53). For both tributaries, however, stream redox potentials we re significantly lower than those measured at the bank and upland (p<0.001, Figure 3-4).

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61 Nutrient Limitation Study Denitrification rates in the ambient and +N sa mples were significantly lower than the +N+C treatment (p= 0.013). Mean denitrifi cation rates were 0.54 0.64 mg kg soil-1d-1 for ambient soils, 1.56 2.68 mg kg soil-1d-1for + N soils, and 7.17 mg kg soil-1d-1 for +N+C (Figure 3-5). In this experiment, there were significant re lationships between deni trification rate and organic matter, moisture content, WEC, and WEN (Table 3-3). When soil samples were compared by location or transect there were no differences in denitrification rates among treatments. Denitrific ation rates were compared between tributaries, however, and rates in T2 were si gnificantly higher than T1 for all treatments (p<0.001; Table 3-4). T1 had an average denitrification rate of 1.51 mg kg soil-1d-1 3.64 whereas T2 had an average of 5.18 9.18 mg kg soil-1d-1. Intact Core Study For all 30 cores, average NO3 removed from the water column was 0.67 0.40 mg L-1d-1. There were no significant differe nces in nitrate removed per day between the floodplain, upland, bank or stream channel (Figure 3-6). One set of cores from the stream channel in the middle transect were outliers and had high rates of denitrification likely due to the presence of worms. Worms can affect denitrification ei ther via gut den itrification or increased sediment water mixing. When cores were analyzed without these soils, the channel soils had significantly lower rates of denitrification than the bank so ils (p=0.04, Table 3-5, Figure 3-6). There were also differences in nitrate removal rate by transect. The middle transect had an average NO3 removal rate of 1.02 0.42 mg L-1 d-1 and was significantly different from the upper transect (0.49 0.39 mg L-1d-1; p= 0.19) but not the lowe r transect (0.66 0.10 mg L-1d-1). The lower and upper transects were not significantly different.

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62 Nitrate removal rate in the core water colu mn was significantly correlated with DEA rate, moisture content and organic matter, but not with other soil parameters (Table 3-6). The soil cores had an average DEA rate of 29.62 42.84 mg kg soil-1 d-1, however, this rate is much higher because it in cludes the floodplain DEA. Wit hout floodplain rates, the mean DEA rate was 16.49 17.94 mg kg soil-1 d-1. DEA rates for the core soils were highly correlated with organic matter and moisture (Figure 3-7 and 3-8). DEA was al so significantly correlated with WEC (r = 0.57), but not WEN (r= 0). Discussion Soil Characterization Study For all soil characteristics measured in T1 and T2, only bulk density and denitrification enzyme activity (DEA) rates were significantly diffe rent. DEA rates were quite variable in both T1 and T2, but this is likely the result of microsites with high carbon or high moisture (Parkin 1987 and Tiedje et al. 1984). DEA rates measured in this system were an order of magnitude lower than those measured by White and Reddy in the Everglad es, Florida (2003). Everglades soils, however, are peat soils that accumulate carbon and receive waters hi gh in nitrogen and phosphorus. Lowrance (1992) measured a mean DEA rate of 0.191 mg kg soil-1d-1 on soils in the Gulf Atlantic coastal plain in Georgia, compared to a mean of 5.89 mg kg soil-1d-1 found in the SFBRU soils. Because DEA is a measure of denitrification potential, the results s uggest that under ideal conditions, higher rates of denitrific ation will occur in T1 compared to T2. This is likely because T1 soils have a steady nitrate source to utilize in the water column, whereas T2 has low nitrate concentrations and thus, denitrif ication is limited by nitrate. Redox potentials measured in the stream channe l also show T1 redox to be in the optimum range for nitrate reduction, whereas in T2, redox potentials are too low for nitrate to be the

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63 dominant electron acceptor. Rates of denitrific ation in T1 suggest the reduction of nitrate observed along the length of the tributary is likely in part due to denitrification. DEA rates were significantly higher in the bank and upland soils of both tributaries compared to soils in the stream channel. Because denitrification rates were highly correlated with organic matter content and water extractable car bon, it is likely that the observed differences in denitrification by stream location are related to carbon availability. T1 upl and and bank soils were significantly higher in carbon than stream channel soils and T2 upland soils were higher in carbon than bank and stream channel soils. Although denitrification potenti als were higher in the upland and bank soils, redox measurements show these soils were using O2 as the dominant electron acceptor and were, therefore, aerobic. These zones would be ideal for denitrification, but on ly when flooded will the soils become anaerobic enough to carry out denitrification. Nutrient Limitation Study The nutrient limitation study showed that deni trification rates were limited by both carbon and nitrogen, but most strongly by carbon. Nitr ogen and carbon, however, might be co-limiting. Fischer and Whalen (2005) measured the effect of the addition of nitrate, glucose, and nitrate + glucose on DEA rates. Highest rates were obtained in the nitrate + glucose treatment, similar to our findings. In their experiment, however, ther e were no significant differences between the nitrate and glucose treatments. Unlike the DEA experiments, soils in T2 had si gnificantly higher denitrif ication rates than in T1. This is likely due to the presence of ch loroamphenicol in the DEA solution, which blocks the microbial production of new enzymes for denitrifi cation, allowing only enzymes already present in the soil to be used for denitrification. T2 has low nitrate availability so it is less likely that microbes are using NO3 as an electron acceptor for respira tion. Depending on how reduced these

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64 soils are, microbes would be using other electron acceptors such as O2, Fe3+ or CH4 during respiration. The nutrient limitation study, however does not use chloroamphenicol, so microbes can produce new enzymes to carry out denitrification. There are likely differences between the tributaries in microbial activity, micronutrients, or soil texture th at are driving differences in denitrification rates with and without chloroampheni col. Studies have shown that soil texture can affect denitrification (Godde and Conrad 2000; Grof fman and Tiejde 1991). DHaene et al. (2003) found highest denitrificati on rates in clay soils (l ow bulk densities) and lowest rates in sandy soils (high bulk densities). This may help explain diffe rences in our findings for the nutrient limitation study since higher rates of denitrif ication were found in T2 soils with lower bulk densities than those in T1. Intact Core Study Intact soil core nitrate removal rates were highly variable, ranging from 0.01 to 1.94 mg L1d-1. When soils were initially compared by samp le location, no significant differences were found. Tubificid worms were observed in a set of cores from the stream channel of the middle transect. Some tubificid worms are able to to lerate low oxygen conditions and often occur in low nutrient conditions (Howmiller 1975). Although cores with worms had extremely low organic matter content, they had the highest net nitr ate removed from the water column during the experiment. Studies have shown that tubificid worms significantly increa se rates of microbial processes because bioturbation allo ws surface particles and chemical species to infiltrate to lower depths in the soil (Mermillod-Blondin et al 2004). This could have increased the NO3 transport rate from the aerobic water column to anaerobic s ites in the soil where denitr ification takes place. When these cores were excluded from analysis mean nitrate removal rates from channel cores were lower than upland soils and significan tly lower than bank soils. This is similar to findings in previous experiments, and is likely due to car bon availability.

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65 Intact core nitrate removal rates can be qu ite different from deni trification potentials measured in the lab. The presence of plants and bioturbators can influence the process of denitrification. Studies have s hown that the presence of plant r oots can increase denitrification rates because nitrification can take place in th e oxygenated zone surrounding roots (Hernandez and Mitsch 2006). Nitrate is then ava ilable to diffuse to surrounding anaer obic zones in saturated soils. This experiment did not remove small plants or plant roots from soil cores due to the soil disturbance it would have caused in the intact cores. All cores w ith plants present removed more nitrate than those without plants but it was unclear if this wa s a result of plant uptake or denitrification. In the future, it would be interesting to compare N2O emission from cores with and without plant roots. The DEA in intact core soils were higher than in previous experime nts. These findings are likely the result of soils in th e intact cores being saturated throughout the experiment, allowing them to become anaerobic for a longer period of time. Under anaerobic conditions, more enzymes would be produced by denitrifying bact eria to carry out denitrificati on in the presence of a nitrate source. The microbial community would be utilizing nitrate as an electron acceptor in the process of denitrification, and nitrat e concentrations in the wate r column would decrease. DEA rates were also highly correlated with por osity in these soils. This may be because larger porosities allow more nitrate to diffuse in to the anaerobic portion of the soil profile. In summary, DEA rates were higher in T1 th an in T2 for the soil characterization study likely due to the lack of denitrification occurring in the low nitrate T2. Denitrification rates were highest in upland and bank soils compared to stream channel soils, likely due to carbon availability. The nutrient limitation study showed that denitrification in both tributaries was limited by nitrate and carbon. T2 denitrification ra tes were higher when soils were incubated for

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66 longer time periods in the absence of chloroamphe nicol possibly due to differences in microbial activity and soils texture. Fina lly, in the intact core study, intact soil core nitrate removal rates were lowest in the stream channel in the absence of tubificid worms. Highest nitrate removal rates were found in ba nk soils indicating that, when flooded, these zones would be optimal for denitrification. DEA rates were also higher in these soils than in previous experiments, likely due to the fact that soils were flooded prev ious to denitrification potential measurements.

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67 Figure 3-1. Sampling sites on each tributary. Each transect had sample stations at the upland (U), bank (B), and stream channel (S). U U B B S U U B B S U U B B S Upper transect Middle transect Lower transect

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68Table 3-1. Soil characteristics in tributar y 1 (T1) and tributary 2 (T2) for the upl and, bank, and stream. Values (n=15) repr esent mean and one standard deviation. Values with different letters indi cate that upland, bank, and stream characteristics are significantly different wi thin each tributary ( =0.05). BD (g cm-3)pHWEN (mg kg-1)WEC (mg kg-1)%OM%Moisture Mean1.23 (0.32) 5.82 (0.98) 1.25 (2.45) 9.33 (5.03) 5.21 (3.67) 18.85 (12.16) Upland mean 1.05 (0.19) a 5.43 (1.14) a2.70 (3.41) a10.62 (3.41) a 7.22 (1.52) a8.36 (2.72) a Bank mean1.24 (0.35) a6.00 (0.75) a0.33 (0.40) b10.54 (6.06) a 5.19 (4.41) a27.50 (12.96) b Stream mean1.58 (0.16) b6.22 (0.87) a0.22 (0.33) b4.32 (1.14) b1.23 (0.93) b22.56 (3.09) b BD (g cm-3)pHWEN (mg kg-1)WEC (mg kg-1)%OM%Moisture Mean1.08 (0.34) 5.55 ( 0.64) 2.45 (4.20) 7.76 (4.78) 6.43 (5.82) 17.30 (12.35) Upland mean0.89 (0.24) a5.13 (0.54) a4.05 (4.79) a11.16 (5.21) a 10.17 (6.26) a 18.70 (16.41) a Bank mean1.06 (0.19) a5.59 (0.40) b0.97 (0.74) b6.38 (2.98) b 4.91 (3.14) b12.94 (8.49) a Stream mean1.58 (0.16) b6.31 (0.50) c0.27 (0.28) b3.72 (0.66) b 1.00 (0.66) b20.29 (2.27) a Tributary 2 Tributary 1

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69 0 5 10 15 20 25 30 UplandBankStreamDEA rate (mg kg soil-1 d -1) T1 T2 Figure 3-2. Mean denitrification enzyme ac tivity (DEA) rates in tributary 1 (T1) a nd tributary 2 (T2) in the upland, bank, and stream. Error bars represent on e standard deviation. B A A p< 0.001

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70Table 3-2. Pearson product moment correla tions (r value) between denitrification enzyme activity (DEA) rates and soil characte ristics for tributary 1 and 2. TributaryBulk DensitypHWENWEC%OM%Moisture 1 DEA rate0.730.350.330.690.800.08 2 DEA rate0.600.070.320.500.700.12 Soil Parameters

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71 Bank Stream Upland -2-1012 Factor 1= 49% of variation -2 -1 0 1 2 Factor 2= 24% of variation Figure 3-3. Factor analysis of soil characteristics and stream location. Moisture content DEA rate WEC Soil nitrate BD Organic matter pH

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72 -400 -200 0 200 400 600 800 UplandBankStream LocationRedox Potential (mV) T1 T2 Figure 3-4. Mean redox potentials in tributary 1 (T1) and tribut ary 2 (T2). Nitrate is the dominant electron acceptor for redox potentials from 200-250mV. Error bars represent one standard deviation. 0 2 4 6 8 10 12 14 16 18 20 AmbientN addedN and C added TreatmentDenitrification rate (mg kg soil-1 d-1) Figure 3-5. Mean + one standard deviation of denitrification rates for each treatment of a nutrient limitation experiment (N= n itrogen, N and C= nitrogen and carbon). A B p< 0.001 A p= 0.013 A A B

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73 Table 3-3. Pearson product moment correlations (r value) between denitrification rates of each treatment and soil characteristics. Soil parameterAmbientN addedC and N added % Organic matter0.500.730.66 % moisture0.260.390.16 WEC0.400.740.55 WEN0.240.560.43 Table 3-4. Mean one standard de viation of denitrification rates for each treatment in tributary 1 (T1) and tributary 2 (T2). TreatmentT1T2 Ambient0.19 0.130.73 0.71 (+) N0.38 0.332.74 3.46 (+) N (+) C3.18 5.3411.17 13.28 mg kg soil-1 d-1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2Floodplain Chann el B a nk U p la nd Chan n el Ban k U p land Channel Bank Contr olLocationNitrate removal rate (mg L-1d-1) Figure 3-6. Mean nitrate removal rates + one standa rd deviation of each set of intact soil cores. The upland of the upper transect was not sa mpled because of equipment problems. Tubificid worms were found in the set of th ree cores taken from the channel of the middle transect. Lower transect Upper transect Middle transect

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74 Table 3-5. Mean nitrate removal rate one stan dard deviation by sampling site. Mean values followed by the same value are not significan tly different. This analysis excludes a set of cores that were outliers. LocationNitrate removal rateSD Upland0.67 ab0.26 Bank0.79 a0.14 Stream0.44 b0.33 mg L-1d-1 Table 3-6. Pearson product moment correlations between nitrate removal rate per day and soil characteristics (DEA is denitrificati on enzyme activity, WEC and WEN are water extractable carbon and water extr actable nitrogen, respectively). Soil Parameterr Organic matter (%)0.38 Moisture content (%)0.36 DEA rate (mg kg-1 d-1)0.47 WEC (mg kg soil-1)0.22 WEN (mg kg soil-1)0.16

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75 y = 1.33x + 0.53 R2 = 0.677 -3 -2 -1 0 1 2 3 4 5 6 0.00.51.01.52.02.53.03.5 Organic Matter (%)DEA rate (mg kg soil-1 d-1) Figure 3-7. Linear relationshi p between organic matter and denitrification enzyme activity (DEA) for the core soils. y = 324.9x 84.3 R2 = 0.868 -50 0 50 100 150 200 0.150.250.350.450.550.650.75 PorosityDEA rate (mg L-1d-1) Figure 3-8. Linear relationship between porosity and denitrification enzyme activity (DEA) rate for core soils.

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76 CHAPTER 4 SUMMARY, IMPLICATIONS AND FUTURE RESEARCH Water Quality Monitoring Many tributaries of the Santa Fe River drain agricultural land in north-central Florida. There are direct connections be tween ground and surface waters in this region as a result of a discontinuous clay layer (the Hawthorne layer) that overlays lim estone bedrock. When water runs off agricultural areas, waters high in ni trogen, phosphorus, and othe r nutrients can impact fresh and marine waters. N itrate-nitrogen can affect the health of humans, animals, and ecosystems, so improvements in management of agricultural runoff are important to maintain Floridas drinking water quality and ecosystem health. Two tributaries of the Santa Fe River were studied for one year. One tributary had consistently low nitrate concentrations, while th e other was high in nitr ate from runoff of an ornamental plant nursery. Our st udy found that nitrate is being at least part ially reduced in the impacted tributary as a result of denitrificati on processes prior to wate r reaching the Santa Fe River. Nitrate concentrations varied over the year of monitori ng likely due to season, fertilizer application rates, a nd irrigation rates. Our results indicate that open water reaches a nd reaches with herbaceous vegetation of this tributary are more efficient at nitrate removal than others. To decrease nitrogen loading to Floridas waters, a number of Best Management Practices (BMPs) could be implemented that create or enhance these open water and herbaceous vegetation reaches in tributaries of the Santa Fe River. For instance, small dams or weirs coul d be installed to decreas e flow or pool water to create open water reaches. To create reaches with herbaceous vegetation, organic and mineral material could be deposited in a tributary. Af ter stabilization, these depositional areas could be planted with native hydroph ilic herbaceous plants.

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77 Soil Characterization and Denitrification Our denitrification experiments showed th at bank and upland so ils had the highest denitrification potentials. Unless the stream ch annel overflows, however, these aerobic soils are not active sites for nitrate removal from the wa ter column. BMPs could be implemented that decrease bank incision to encourage water to over flow banks or increase contact with riparian and upland soils. Otherwise, grea test denitrification will only o ccur in these soils during storm events. To increase denitrification occurring in th e stream channel, wher e our findings suggest carbon is limiting, adding a carbon substrate of so me sort may be feasible. One such BMP employs a denitrification wall that is constructe d in water systems to improve nitrate removal. These walls have been shown to greatly impr ove nitrate removal capacity by providing a carbon source for the process of denitr ification (Greenan et al 2006). These walls are long-lasting, inexpensive, and easy to insta ll. Common carbon sources are saw dust, peanut shells, wood chips and plant residues. In fact, a de nitrification wall is planned for installation upstream of T1 in conjunction with the plant nurser y. Schipper and Vojvodic-Vukovic (2001) found that after five years, a denitrification wall had the same perf ormance, and only when the water table dropped below the wall did nitrate concentrations downstream increase. The nutrient limitation study indicated th at nitrate and carbon are both limiting denitrification in these soils. The intact core st udy also indicated that denitrification is limited by carbon, and low oxygen, anaerobic conditions. Sa turation of upland and bank soils could significantly increase denitrification in this syst em and improve nitrate re moval from tributaries in the Santa Fe River. Intact soil core nitrate removal rates were more variable than DEA rates measured in the soils likely because intact soil co res are more representative of field conditions.

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78 Future Research This research focused on two tributaries in the Santa Fe River Watershed. A study that monitors multiple tributaries across the watershed would be helpful to address if processes across tributaries are similar. A number of BMPs coul d then be implemented and tested for success to decrease the impact that agricultural areas have on the Santa Fe River and other freshwater systems in the Santa Fe River watershed. More research is also needed to understand what role plants have in removing nitrate from reaches of these tributaries. Plants do not provi de a long-term sink for nitrogen and, thus, if they are removing nitrogen from the system, it may be helpful to harvest the plants to prevent the release of stored nitrogen back into the tributaries. Soil samples from different tributary reaches could also be tested for denitrification potentials. This would increase und erstanding of the role of denitr ification in individual reaches within the stream channel. To increase nitr ate removal, construction of similar reaches along tributaries, or increasing the residence time of water in these tributarie s could greatly increase nitrate removal efficiencies. For instance, in this study, Open Water and Depositional Herbaceous reaches removed the most nitrate from the water column. Open Water reaches can be created in a tributary by narrowing the tributary channel downstream from a reach. Depositional Herbaceous reaches can be constructed by depositing materials within the stream that will recruit plants and force a sha llow water column to flow over the reach. The floodplain was shown to reduce both nitrate and SRP concentrations in T1, and floodplain soils had high DEA rates. More resear ch would be useful to study the importance of

PAGE 79

79 the floodplain in reducing nutrients in agriculture runoff. It may be beneficial to restore abandoned pastures and other land to floodplain to reduce N and P loading to the Santa Fe River. Conclusion This study provided evidence that a tributar y of the Santa Fe River reduces nitrate concentrations in agricultural runoff. Denitrif ication is believed to be a major process reducing nitrate concentrations, though a combination of carbon, nitrogen, and saturated anaerobic soils are limiting denitrification. BMPs such as deni trification walls and mo rphological stream reach enhancements are suggested to increase nitrate rem oval from tributaries near agriculture areas. These BMPs, however, could alter stream ecosyst em function, so, it would be ideal to manage agriculture runoff before it enters water systems. This involves reducing fertilizer applications, intercepting runoff with buffer st rips, or controlling on-site dr ainage (Hey 2002). Reducing or optimizing fertilizer applications would also d ecrease greenhouse gases produced as by-products of denitrification. There is curr ently little incentive fo r agriculture, industry or municipalities to regulate nitrates.

PAGE 80

80 LIST OF REFERENCES Aber, JD, Magill, A, McNulty, SG, Boone, RD, Nadelhoffer, KJ, Downs, and M, Hallett, R. 1995. Forest biogeochemistry and primary production altered by nitrogen saturation. Water, Air and Soil Pollution 85: 1665. Aulakh, MS, Doran, JW, Walters, DT and Power, JF. 1991. Legume residue and soils water effects on denitrificati on in soils of different text ures. Soil Biol. Biochem. 23: 1161 Bledsoe, EL, Phlips, EJ, Jett, CE, and D onnelly, KA. 2004. The relationships among phytoplankton biomass, nutri ent loading, and hydrodynami cs in an inner-shelf estuary. Ophelia 58(1): 29. Bowden, W.B. 1986. Gaseous nitrogen emissions from undisturbed terrestrial ecosystems. An assessment of their impact s on local and global nitrogen budgets. Biogeochemistry 2:2492 Brady, NC, and Weil, RR. 2002. The Nature and Properties of Soils. 13 ed. P. 544, 554. Burns, I.G. 1992. Influence of plant nutrient con centration on growth-rate. Use of a Nutrient interruption technique to determine critical concentrations O, N, P and K in young plants. Plant and soil 142(2): 221 Capblancq, J. 1999. Nutrient dynamics and pelagic food web interactions in oligotrophic and eutrophic environmentsan overview. Hydrobiologia 207:1 Chapin III, F.S., Matson, P.A., and Mooney, H.A. 2004. Principles of Ecosystem Ecology. Principles of Ecosystem Ecology. 1 ed. Springer, New York, NY. DAngelo, E.M., and K.R. Reddy. 1999. Regulators of heterotrophic microbial potentials in wetland soils. Soil Biology and Biochemistry 31:815 Desimone, Leslie A. and Howes, Brian L. 1996. Denitrification and Nitrogen transport in a Coastal Aquifer Receiving Wastewater Discharge. Environmental Science and Technology 30: 1152 D'Haene K, Moreels E, De Neve S, Daguilar BC, Boeckx P, Hofman G, and Van Cleemput O. 2003. Soil properties influencing the denitrif ication potential of Flemish agricultural soils. Biology and fertility of soils 38 (6): 358 Diaz, RJ. 2001. Overview of Hypoxia around th e World. Journal of Environmental Quality 30:275 Fennessy MS, and Cronk JK.. 1997. The effectiveness and re storation potential of riparian ecotones for the management of nonpoint source pollution, particularly nitrate Critical Reviews in Environmen tal science and technology 27 (4): 285

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81 Follett JR, Follett RF. 2001. Utilizati on and metabolism of nitrogen by humans. Pages 65 in Follett R, Hatfield JL, eds. Nitrogen in the Environment: Sources, Problems and Ma nagement. Amsterdam (Netherlands): Elsevier Science. Forman, D. 2004. Commentary: Nitr ites, nitrates and nitrosation as causes of brain cancer in children: epidemiological ch allenges. International J ournal of Epidemiology 33(6): 1216. Galloway, JN, Aber, JD, Erisman, JW, Seitzinger, SP, Howarth, RW, Cowling, EB, and Cosby, J. 2003. The Nitrogen Cascade. Bioscience Vol 53(4): 341. Galloway JN, Dentener FJ, Capone DG, Boyer EW, Howarth RW, Seitzinger SP, Asner GP, Cleveland CC, Green PA, Holland EA, Karl DM, Michaels AF, Porter JH, Townsend AR, Vorosmarty CJ. 2004. Nitrogen cycles: past, present and future. Biogeochemistr y 70 (2). Goldman, J.C. 1999. Identification of nitrogen as a growth-limiting nutrient in wastewaters and coastal marine waters through continuous culture al gal assays, Water Research, Volume 10, Issue 2, 1976, Pages 97. Godde, M and Conrand, R. 2000. Influence of soil properties on the turnove r of nitric oxide and nitrous oxide by nitrification a nd denitrification at constant temperature and moisture. Biology and fertility of soils 32 (2): 120. Greenan, CM, Moorman, TB, Kaspar, TC, Park in, TB, and Jaynes, DB. 2006. Comparing carbon substrates for denitrification of s ubsurface drainage water. Journal of Environmental Quality 35: 824. Groffman, P.M., and Tiedje, JM. 1991. Re lationships between denitrification, [CO2] production and air-filled porosity in soils of differe nt texture and draina ge. Soil Biology and Biochemistry 23:299. Hanlon, E.A. 1984. IFAS Extension Soil Testi ng Laboratory Chemical Procedure and Training manuel. IFAS University of Florida, Gainesville, Florida. Hefting, M., Clement, JC, Dowrick, D, Cosandey, AC, Bernal, S, Cimpian, C., Tatur, A, Burt, TP, and Pinay, G. 2004. Biogeochemistry 67: 113. Hernandez, ME, and Mitsch, WJ. 2006. Influe nce of hydrologic pulses, flooding frequency, and vegetation on nitrous oxide emissions from created riparian marshes. Wetlands 26(3) 862. Howmiller, RP. 1975. On the Abundance of Tubi ficidae (Annelida: Oligochaeta) in the profundal bethos of some Wisconsin lakes. The American Midland Naturalist 97 (1) 211.

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82 Jackson, M.L. 1993. Soil Chemical Analysis. Prentice Hall, Inc., Englewood Cliffs, NJ., EPA. Methods for the determination of inorganic substances in environmental samples. 365.1. Johns D, Williams H, Farrish K, and Wagner, S. 2004. Denitrification and soil characteristics of wetlands created on two mine soils in east Texas, USA Wetlands 24 (1): 57. Lowrance, R. 1992. Groundwater Nitr ate and Denitrification in a Co astal Plain Riparian Forest. Journal of Environmental Quality 21(3): 401. Lowrance, R., Johnson, J.C., Newton, G.L., and Willia ms, R.G. 1998. Denitrification from soils of a year-round forage production system fe rtilized with liquid manure. Journal of Environmental Quality 27 (6): 1504. Maag, M, Malinovsky, M., and Nielson, SM. 1997. Kinetics and temperature dependence of potential denitrification in riparian soils. Journal of Environmental Quality 26:21523. Martienssen, M., and Schops, R. 1999. Populati on Dynamics of Denitrifying Bacteria in a model biocommunity. Water Res. 33: 639. Martin, J, Screaton, E., and Moore, P.2004. Surface and ground water mixing along the Cody Scarp: An example from the Santa Fe Rive r Sink-Rise system. USGS Suwannee River Basin and Estuary Integrated Scienc e Workshop Proceedings, September 22. Mermillod-Blondin, F, Gaudet, JP, Gerino, M., Desrosiers, G., Jose, J., and Creuze des Chatelliers, M. 2004. Fr eshwater Biology 49: 895. Murray, RE and Knowles, R. 1999. Chloroamph enicol Inhibition of Denitrifying Enzyme Activity in two agricultural soils. App lied and Environmental Microbiology 65(8): 3487. Parkin, TB. 1987. Soil microsites as a source of de nitrification variabilit y. Soil Science Society of America 51: 1194. Phlips, EJ, Badylak, S, and Grosskopf, T. 2002. Factors affecting the abundance of phytoplankton in a restricted subtropical lagoon, the Indian River Lagoon, FL, USA. Estuarine, Coastal, and Shelf Science 55(3): 385. Nolan, BT and Stone, JD. 2000. Nutrients in Groundwaters of the Conterminous United States, 1992-1995. Environmental Scien ce and Technology (34), 1156. Rabalais, NN, Wiseman, WJ, Turner, RE, SenGupta BK, Dortch, Q. 1996. Nutrient changes in the Mississippi River and system responses on the adjacent continental shelf. Estuaries 19 (2B): 386.

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83 Reddy, KR, Patrick, WH, and Lindau, CW. 1989. Nitrification-denitr ification at the plant rootsediment interface in wetlands. Limnology and Oceanography 1004. Rosgen, D.L. and H.L. Silvey. 1996. Applied River Morphology. Wildland Hydrology Books, Fort Collins, CO. Rouse, DR, Bishop, CA and Struger, J. 1999. Nitrogen pollution: an assessment of its threat to amphibian survival. Environmen tal Health Pers pectives 107: 799. Schaede, JD and Lewis, DB. 2006. Plastici ty in Resource Allocation and Nitrogen-use Efficiency in Riparian Vegetation: Implications for Nitrogen Retention 9:740. Schipper, LA and Vojvodic-Vukovic, M. 2001. Five years of nitrate rem oval, denitrification and carbon dynamics in a denitrificati on wall. Water Research 35(14): 3473. Schipper, L.A., Cooper, A.B., Harf oot, C.G. and Dyck, W.J. 1993. Regulators of denitrification in an organic riparian soil. Soil Biology and Biogeochemistry 25 (7): 925. Schlesinger, W.H. 1997. Bioge ochemistry, An Analysis of Global Change. Academic Press Second Edition. p394. Schnabel, Ronald R., Shaffer, John A., Stout, William L., and Cornish, Leonard F. 1997. Denitrification in Four Valley and Ridge Riparian Ecosystems. Environmental Management 21: 283. Seitzinger, S.P. 1988. Denitrification in freshwater and coastal marine ecosystemsecological and geochemical significance. Limnol ogy and Oceanography 33 (4): 702 Part 2. Sirivedhin, T. and Gray, KA. 2006. Ecological Engineering. F actors affecting denitrification rates in experimental wetlands: Field and laboratory studies. 26: 167. Smialek, J, Bouchard, V, Lippmann, B, Quigley, M, Granata, T, Martin, J, and Brown, L. 2006. Effect of a woody ( Salix nigra ) and an herbaceous ( Juncus effuses ) macrophyte species on methane dynamics and denitrification. Wetlands 26(7): 509. Strauss, EA and Lamberti, GA. 2002. Effect of dissolved organic carbon quality on microbial decomposition and nitrification rates in str eam sediments. Freshwater Biology (47): 65 74. The Florida Speological Society 2004. h ttp://www.caves.com/fss/pages/misc/geology.htm Thomas, G.W. 1996. Soil pH and Soil Acidity. In Methods of Soil Analysis. Part 3. Chemical Methods. P. 475. J.M. Bigham, (ed.) Soil Science Society of America, ASA, Madison, Wisconsin.

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84 Tiedje, JM, Sexstone, AJ, Parkin, TB, Revs bech, NP, and Shelton, DR. 1984. Anaerobic processes in soil. Plant Soil 76: 197. van der Hoek, Dick, van Mierlo, Anita J.E.M ., van Groenendael, Jan M. 2004. Nutrient limitation and nutrient-driven shifts in plan t species composition in a species-rich fen meadow. Journal of Vegetation Science 15: 389. Vitousek PM,Howarth RW, Likens GE,M atson PA, Schindler D, Schlesinger WH, Tilman GD. 1997. Human alteration of the global nitrogen cycle: Causes and consequences. Issues in Ecology 1: 1.

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85 BIOGRAPHICAL SKETCH Adrienne Elizabeth Frisbee was born Septem ber 23, 1979, in Dallas, Texas. She grew up in Tulsa, Oklahoma and discovered her love of science early in biology class at Bishop Kelley High School. When she graduated in 1998, she m oved to New Orleans, Louisiana to attend Loyola University. There, she majored in biolog y with a minor in environmental studies. She graduated cum laude in 2002. For the next two years, Adrienne was a biol ogist in New York, Ca lifornia, and Oklahoma studying vegetation restoration a nd coastal and grassland bird species. After deciding to continue her education, she moved to Florida in 2004 to get her Ma sters of Science degree in soil and water science. There she studied wetlands and water quality and wa s also involved in research projects in Alaska. After graduating in May 2007, Adrienne will be working in San Francisco, CA with NASA on nitrogen cycling in microbial mats.


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Title: Nitrate-Nitrogen Dynamics in Tributaries of the Santa Fe River Watershed, North-Central Florida
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Material Information

Title: Nitrate-Nitrogen Dynamics in Tributaries of the Santa Fe River Watershed, North-Central Florida
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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NITRATE-NITROGEN DYNAMICS IN TRIBUTARIES OF THE SANTA FE RIVER
WATERSHED, NORTH-CENTRAL FLORIDA




















By

ADRIENNE E. FRISBEE


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

2007




































O 2007 Adrienne E Frisbee



























I would like to thank"Auntie"Adele Szablowski, who helped instill in me the belief that I could
do this; my father, Joe Frisbee, whose unconditional love and support through all of my j ourneys
continues to amaze me; and my sister, Jeanine Firmin, my very best friend through it all.









ACKNOWLEDGMENTS

I would like to thank my advisor Mark Clark for his guidance and support, as well as my

committee members, Richard Lowrance, Michelle Mack, and K.R.Reddy, and for their input on

this proj ect.

I thank everyone in the Wetland Biogeochemistry laboratory, especially Yu Wang and

Gavin Wilson for invaluable training with methods and instrumentation. I wish to thank Isabella

Claret Torres for her input on sampling, methods, and statistics, as well as her support as a

friend. I would like to thank Ed Dunne, Angelique Keppler, and Kanika Inglett for their input as

well. I would also like to thank Jenny Schafer for her immense help with editing. I would like to

thank Jason Smith for many helpful biogeochemistry talks and for helping me get a job in

California.

I would like to thank the University of Florida Department of Animals Sciences and the

Suwannee River Water Management District for financial support.

Finally, I would like to thank my friends and family for their love and support. I am

especially grateful to Hanna Lee, Melissa Lott and Jenny Schafer for their tremendous support.












TABLE OF CONTENTS


page


ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............7............ ....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 BACKGROUND AND SITE DESCRIPTION ................. ....._._ ......._ ...........1


Introducti on ................... ......_ ._ ....... .. .....__..........1

Nitrogen Removal in Riparian Wetlands............... ...............14
Regulators of Denitrification ................. ...............15........_......
Nitrogen and Florida' s Waters.. ..........._.._ ............_ ......... ......_ ....._..__...18
The Santa Fe River Watershed .............. ...............19....

Obj ectives and Hypotheses ........._.__....... .__. ...............20...


2 WATER QUALITY MONITORING ................. ...............25................


Introducti on .................. ........... ...............25.......

Obj ectives and Hypotheses ................. ...............26................
Materials and Methods .............. ...............26....
Site Description .............. ...............26....
Field Methods ................. ...............27.................

Analytical M ethods .............. ...............28....
Statistical M ethods .............. ...............29....
Re sults ................ ...............29.................
N itrate ............... ... ............ ...............29.......
Ammonium and Organic Nitrogen ............... ... ........... .. ...............30.....
Dissolved Organic Carbon and Soluble Reactive Phosphorus ................. ................ ..30
Chloride ................. ...............3.. 1..............

Floodplain ................. ...............3.. 1..............
Nitrate ................. ...............3.. 1..............

Phosphate .............. ...............3 1....
Discussion ................. ...............3.. 1..............


3 SOIL CHARACTERIZATION AND DENITRIFICATION ................. ........... ...........52


Introducti on ................. ...............52._ ___.......

Obj ectives ............. ........... ...............52....
Hypotheses .............. ...............53....
Materials and Methods .............. ...............53....













Sampling Locations ................. ...............53.................
Soil Characterization Study ................. ...............53................
Field m ethods .............. ...............53....
Laboratory methods............... ...............54
Denitrification potential .............. ...............55....
Nutrient Limitation Study............... ...............56.
Field m ethods .............. ...............56....
Laboratory methods............... ...............56
Intact Core Study ................. ...............57........... ....
Field m ethods .............. ...............57....

Laboratory methods............... ...............58
Stati sti cs ........._.__....... .__ ...............58...
Re sults........._. _....... _._ _._ .. ..... ...............59
Soil Characterization Study ........._.__....... .__. ...............59...
Nutrient Limitation Study............... ...............61.
Intact Core Study ................. ...............61........... ....
D discussion ................... ............ ...............62.......
Soil Characterization Study ................. ...............62................
Nutrient Limitation Study............... ...............63.
Intact Core Study ................. ...............64........... ....


4 SUMMARY, IMPLICATIONS AND FUTURE RESEARCH .............. ....................7


Water Quality Monitoring .................. ...............76..
Soil Characterization and Denitrification ................. ....._.. ...............77. ....
Future Research .............. ...............78....
Conclusion ....._._ ................ ........_.. .........79


LI ST OF REFERENCE S ..........._.._. .......... ...............8_ 0...


BIOGRAPHICAL SKETCH .............. ...............85....










LIST OF TABLES


Table page

2-2 Average percent change in nitrate per meter in tributary 1 (T1) for 11 months.
Values with the same letter for significance level (SL) are not significantly different.....44

2-3 Summary of NH4' and TKN measured in tributary (T1) and tributary 2 (T2) with one
standard deviation in parentheses. Dashes indicate months that were not analyzed
for NH4+ Or TKN ................. ...............47................

3-1 Soil characteristics in tributary 1 (T1) and tributary 2 (T2) for the upland, bank, and
stream. Values (n=15) represent mean and a one standard deviation. ............. ................68

3-2 Pearson product moment correlations (r value) between denitrifieation enzyme
activity (DEA) rates and soil characteristics for tributary 1 and 2. ............. ..................70

3-3 Pearson product moment correlations (r value) between denitrication rates of each
treatment and soil characteristics. .............. ...............73....

3-4 Mean & one standard deviation of denitrifieation rates for each treatment in tributary
1 (T1) and tributary 2 (T2) ................. ...............73........... ..

3-5 Mean nitrate removal rate 1 one standard deviation by sampling site. Mean values
followed by the same value are not significantly different. This analysis excludes a
set of cores that were outliers............... ...............74

3-6 Pearson product moment correlations between nitrate removal rate per day and soil
characteristics (DEA is denitrifieation enzyme activity, WEC and WEN are water
extractable carbon and water extractable nitrogen, respectively). .................. ...............74










LIST OF FIGURES


Figure page

1-1 Along the Ocala Uplift in eastern Florida, the Hawthorne layer becomes
discontinuous, allowing surface and groundwater connections to occur. ................... .......21

1-2 Location of the Santa Fe River Watershed in the Suwannee basin, also represented is
the Cody Scarp (figure used with permission of J. Martin). ................ ............ .........22

1-3 Dominant land uses common in the Santa Fe River Watershed ............... ............._..23

1-4 A digital elevation map of the Santa Fe River Watershed that shows the repeating
pattern of tributaries and where the river goes underground in the western portion of
the watershed .............. ...............24....

2-1 Santa Fe River Beef Research Unit relative to Gainesville, Florida and the Santa Fe
River watershed. ............. ...............36.....

2-2 The SFBRU cattle pastures with an ornamental plant nursery south of the property
and tributaries that drain to the Santa Fe River floodplain. ............. .....................3

2-3 An example of Depositional Woody stream reach. ..........._ ..... .__ ............. ...39

2-4 A Depositional Herbaceous reach. ........._.._.._ ...._.._....._._ ....... ....3

2-5 An example of a Slightly Incised Woody reach. ............. ...............39.....

2-6 Slightly Incised Herbaceous .............. ...............40....

2-7 Deeply Incised Woody............... ...............40.

2-8 An example of an Open Water reach ........._._.._......_.. ...............40...

2-9 A Moderately Incised Woody reach. ............. ...............41.....

2-10 The Santa Fe River floodplain. ............. ...............41.....

2-11 Log of mean nitrate concentrations of tributary 1 (T1) compared to tributary 2 (T2).
Bars represent the standard deviation. T2 was not sampled in October due to the
absence of surface water. .............. ...............42....

2-12 Average and range of nitrate concentration in tributary 1 (T1) from headwaters to
discharge for all months sampled. ............. ...............43.....

2-13 Quantiles of monthly nitrate concentrations measured in T1. ................... ...............4










2-14 Nitrate concentrations by season in tributary 1 (T1). Spring = March, April, May;
summer = June, August; fall = September, October, November; and winter =
January, February................ ...............4

2-15 SRP concentrations in tributary 1 (T1), March 29, 2006............... ...............48..

2-16 SRP concentrations for tributary 2 (T2), March 29, 2006. ............. .....................4

2-17 Chloride concentrations for May 20, 2005 along the length of tributary 1 (T1)............_...49

2-18 Chloride concentrations for May 20, 2005 along the length of tributary (T2). .................49

2-19 Change in nitrate concentrations as tributary 1 (T1) flows through the floodplain and
improved pasture to the Santa Fe River. .....__.....___ ..........__ ..........5

2-20 Change in soluble reactive phosphorus (SRP) concentrations from the last tributary 1
(T1) sample station, through the floodplain and improved pasture to the Santa Fe
River. ........... ..... ._ ...............51...

3-1 Sampling sites on each tributary. Each transect had sample stations at the upland
(U), bank (B), and stream channel (S). ............. ...............67.....

3-2 Mean denitrification enzyme activity (DEA) rates in tributary 1 (T1) and tributary 2
(T2) in the upland, bank, and stream. Error bars represent one standard deviation. .........69

3-3 Factor analysis of soil characteristics and stream location. ............... ...................7

3-4 Mean redox potentials in tributary 1 (T1) and tributary 2 (T2). Nitrate is the
dominant electron acceptor for redox potentials from 200-250mV. Error bars
represent one standard deviation. ........._ ............ ...............72....

3-5 Mean + one standard deviation of denitrification rates for each treatment of a nutrient
limitation experiment (N= nitrogen, N and C= nitrogen and carbon). ............. ................72

3-6 Mean nitrate removal rates + one standard deviation of each set of intact soil cores.
The upland of the upper transect was not sampled because of equipment problems.
Tubificid worms were found in the set of three cores taken from the channel of the
m iddle transect. .............. ...............73....

3-7 Linear relationship between organic matter and denitrification enzyme activity
(DEA) for the core soils. .............. ...............75....

3-8 Linear relationship between porosity and denitrification enzyme activity (DEA) rate
for core soils ................. ...............75........... ....









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

NITRATE-NITROGEN DYNAMICS INT THE TRIBUTARIES OF THE SANTA FE RIVER
WATERSHED, NORTH CENTRAL, FLORIDA

By

Adrienne E. Frisbee

May 2007

Chair: Mark Clark
Major: Soil and Water Science

Nitrate runoff from agricultural systems is an increasing concern because of its potential

effect on the health of both humans and ecosystems. Riparian systems have been shown to

reduce nitrate concentrations in soil and water as a result of denitrification processes that occur

under anaerobic conditions. The Santa Fe River Basin in north central Florida contains many

tributaries that drain adj acent agricultural systems and in the eastern part of the watershed that

discharge to the Santa Fe or New Rivers. In central areas of the Santa Fe River, however, these

tributaries on occasion discharge directly to the Floridian aquifer due to the karst and partially

confined geology of the region. Increasing evidence suggest that nitrate concentrations in surface

and groundwater are increasing, and in some instances have exceeded EPA safe drinking water

standards. In an effort to better understand nitrate dynamics and denitrification potential of

channel bed and riparian wetlands along tributaries of the Santa Fe River, a two year research

investigation was established at Boston Farm-UF/IFAS Santa Fe River Beef Research Unit

(SFRBRU).

Fundamental questions addressed by this research include 1) what are the seasonal

dynamics of nitrate concentrations within two tributaries of the Santa Fe River, 2) are there

differences in stream reach or stream fluvial morphology that influence nitrate assimilative










capacity, 3) what effect does distance from stream have on soil denitrification potential and 4)

what effect does nitrate concentration have on denitrification potential within stream reaches. To

answer these questions two streams on the SFBRU were monitored.

Results show little variation in nitrate concentration along a low nitrate concentration

tributary. Along a high nitrate tributary, however, concentrations were reduced an average of

31% from headwaters to discharge during the study. Decreases in nitrate concentration were not

uniform along the length of the stream, but instead indicate that several types of stream reaches

have significantly greater nitrate assimilative capacities than others.

Soil characterization and denitrification studies indicate that nitrate, carbon and anaerobic

conditions are limiting denitrification in these tributaries.









CHAPTER 1
BACKGROUND AND SITE DESCRIPTION

Introduction

Nitrogen is the most abundant element on earth, yet it is often the most limiting nutrient for

plants and microbes in marine and terrestrial ecosystems (Goldman 1999, Casblanq 1999, Burns

1992). Nitrogen is limiting because it is predominantly present in the atmosphere as dinitrogen

gas (N2), a nitrogen form unavailable to most organisms. Humans, however, have dramatically

increased the amount of available N on earth by a factor of 10 through anthropogenic and

industrial N fixation (Galloway et al. 2004).

In 1913, the Haber-Bosch process was developed to convert N2 to NH3 for fertilizer to

improve food production. Combustion of fossil fuels along with the cultivation of rice, legumes,

and other N-fixing crops has also increased biologically available forms of N, commonly in the

form of ammonium (NH4 ) Or nitrate (NO3-) (Galloway et al. 2004). Inputs of available nitrogen

dramatically increase plant productivity; however, with extensive nitrogen loading to an

ecosystem, more N may be available than plants and microbes can use (Aber et al. 1989). As

excess nitrogen accumulates over time, it can have significant effects within an ecosystem. The

Nitrogen Cascade refers to changes that occur as an ecosystem becomes saturated with nitrogen.

There is an initial increase in productivity; however, over time, nitrogen loading has been shown

to decrease biodiversity in forests, grasslands, lakes, and streams (Aber et al. 1995, Vitousek et

al. 1997). Soil acidicification and a decrease in soil fertility may also occur because leaching of

nitrate ions from the soil facilitates the release of base cations such as calcium.

There are a number of other detrimental effects that nitrogen accumulation can have on the

growth and health of plants in natural and agroecosystems. For instance, with an oversupply of

nitrogen, excessive vegetative growth and plant cell enlargement can cause a plant to become









weak and top heavy. Other effects include delayed plant maturity and reduced resistance to

disease and pests (Brady et al. 2002).

Excess nitrogen loading also has profound effects on waterways. As streams, creeks, or

rivers with elevated levels of dissolved organic nitrogen (DON), ammonium (NH4 ) Or nitrate

(NO3-) drain into ponds, lakes, and oceans, eutrophication and degradation of water quality can

occur (Seitzinger 1988). This can lead to algal blooms, fish kills, change in species composition,

and hypoxic conditions (Rabalais et al. 1996, van der Hoek 2004). Many zones of severe

hypoxia occur where freshwater rivers high in nutrients enter coastal waters such as those near

Louisiana, New York, New Jersey, Alabama, Texas, and Florida leading to mass mortality of

benthic communities and stressed fisheries (Diaz 2001).

Nitrate, an inorganic form of nitrogen, is unique because it is a negatively charged ion,

making it more susceptible to leaching than other positively charged nitrogen species that adhere

to negatively charged soil particles. As nitrate moves in water through the soil and enters ground

and surface waters, it can have detrimental effects on humans, animals, and ecosystems.

Concentrations of nitrate in drinking water greater than 10 mg L^1 are considered a health hazard

to humans and animals. Excess NO3~ Can CaUSe methemoglobinemia or blue baby's syndrome

and has also been linked to brain tumors in children and to forms of stomach cancer (Forman

2004).

Respiratory infections and problems related to thyroid metabolism are also effects

associated with high nitrate levels in drinking water (Follett and Follett 2001). Nitrate

concentrations above 1 mg L-1 have also been shown to be toxic to amphibians and insects

(Rouse et al. 1999).









Nitrogen Removal in Riparian Wetlands

Intact riparian ecosystems have been found to reduce nitrogen concentrations in surface

and groundwater. The ability of these buffer areas to transform nutrients is important in streams

adj acent to agriculture areas that drain to freshwater and marine systems subj ect to

eutrophication (Lowrance 1992). Although these wetlands can be relatively small in area, they

can be a maj or zone for nitrogen retention in plants (Schaede and Lewis 2006) or nitrogen

transformation through denitrification (Fennessy and Cronk 1997).

If ground or surface water comes into contact with plant roots, riparian plants can take up

nutrients from the water column or soil porewater, thus providing a temporary sink for nitrogen.

Schaede and Lewis (2006) found that increased N loading in a nitrogen limited system caused an

increase in plant tissue %/N and changes in the root to shoot ratio in plants due to increases in

nutrient use efficiency and productivity. Yet, as plants senesce, most of the nitrogen will leach

from the plant or be mineralized by microbes, releasing it to the ecosystem. Plants can remove a

significant amount of nitrogen from soil and water; thus, unless plants are harvested or a portion

of the biomass accumulates as peat, they do not provide a long term sink for nitrogen.

Denitrification, another process that removes nitrogen in riparian areas, takes place in soils

and sediments under anaerobic conditions. This reaction occurs when facultative heterotrophic

bacteria must use alternate electron acceptors during respiration under low oxygen conditions.

Nitrate is reduced to dinitrogen, nitric and nitrous oxide gases that are lost to the atmosphere and

thus nitrogen is removed from the water column.

This is a long-term sink for nitrogen since these gases are only available to a few micro-

organisms during nitrogen fixation. As a result of flooded conditions, at least half of the

denitrification on land has been found in wetlands, lake sediments, and riparian ecosystems

(Bowden 1986).










Restored wetlands in agricultural landscapes can be self-sustaining and effective at

removing excess N if properly managed. For the management of animal waste, denitrifieation in

riparian areas can be a valuable process to remove N from liquid manure and other non-point

source pollutants that are land applied (Lowrance et al. 1998). Cleaner water, however, may

come at a cost to air quality. Denitrifieation plays a role in global climate change because it

generates greenhouse gases. IfNO3~ is not reduced completely to N2, miCTObial by-products N20

and NOx will be the end products of denitrifieation. Production of N20 rather than N2 is favored

at low pH (Johns et al. 2004), low temperature, and high oxygen and nitrate concentrations

(Chapin et al. 2002). N20 has a long residence time in the atmosphere due to its low reactivity.

This gas contributes to global warming since it can absorb infrared radiation and has the capacity

to contribute about 300 times the greenhouse effect as one molecule of CO2 (Schlesinger 1997).

Also, in reactions in the stratosphere, this produces NO, a gas that contributes to the destruction

of good ozone.

Another intermediate product of denitrifieation is NOx. This is a very reactive gas that is

involved in the production of stratospheric ozone, or the photochemical smog that is common in

highly populated urban areas. Smog is known to cause lung problems in humans. NOx is also a

component of acid rain in the form of nitric acid. Not only is this a strong acid that decreases the

pH of soils, it also deposits available N in ecosystems.

Regulators of Denitrification

Several factors influence where and at what rate denitrifieation occurs. Denitrification

requires the presence of a labile carbon source, anaerobic conditions, a nitrate source, and an

active microbial community. Other abiotic factors such as temperature and soil texture can

affect rates of denitrifieation.









A number of studies have found the presence of a readily available carbon source to be the

primary factor affecting rates of denitrifieation in ecosystems (Marienssen and Schops 1999).

Under waterlogged conditions, the breakdown of organic matter is slow because, in the absence

of Ol, miCTObes must use an alternate electron acceptor such as CH4, NO3-, Fe3+, Mn 4+, Or SO42-

during respiration. These electron acceptors are not as energetically efficient as 02, which leads

to a slower decomposition rate and the accumulation of organic matter and, thus, electron donors

for the denitrification process (D'Angelo and Reddy 1999). Dissolved organic carbon (DOC),

another source of carbon in riparian ecosystems, has been shown to be highly correlated with

rates of denitrification (Desimone and Howes 1996).

Moisture content also affects denitrification since anaerobic conditions must be present for

denitrification to occur. Studies show that denitrification rates have a significant relationship

with moisture content (Schnabel et al. 1997, Schipper et al. 1993). Schnabel et al. (1997) found

that moisture content decreased with distance from streams in riparian areas and increased with

soil depth; however, where moisture conditions are optimal, other factors such as carbon may be

limited. Moisture content in soils is also affected by water table fluctuations, therefore seasonal

or event driven changes in water table can strongly influence the nitrogen cycle in the processes

of nitrification, mineralization, and denitrification (Reddy et al. 1989). In areas subject to high

loads of nitrogen, a fluctuating water table will increase the nitrogen removal efficiency in

riparian zones (Hefting et al. 2004).

The process of denitrification is also controlled by the presence of a nitrate source.

Systems that are flooded year-round must rely on the diffusion of nitrate from aerobic to

anaerobic layers for denitrification to take place. If a system does not have a nitrate source, then

denitrification may be controlled by nitrification rates. Nitrification is the process where NH4+ is









oxidized to NO3- by autotrophic bacteria. This occurs in aerobic portions of the soil, and nitrate

can diffuse to anaerobic soils along a concentration gradient. Even if microsites within the soil

are anaerobic, seasonal water table fluctuations stimulate nitrification (Schipper et al. 1993). If

02 COncentrations are too low and nitrification cannot occur, however, NO3~ prOduction can be a

rate-limiting step of denitrification.

Denitrification is indirectly affected by soil texture. Higher rates of denitrification have

been found in fine-textured soils rather than sandy soils (Hefting et al. 2004). For instance,

during storm events, flooded conditions ideal for denitrification can be short-lived because of the

rapid drainage that occurs in coarse, sandy soils. This is related to water filled pore space

(WFPS); as WFPS increases so do rates of denitrification. Aulakh et al. (1992) found that

denitrification only occurs at a WFPS of 60% and higher.

All microbial processes are regulated by temperature. Qlo is the rule of thumb that with

every 10 degree increase in temperature, biological activity will double. Denitrification is a

mechanism carried out by microbes, and it has also been shown to be highly affected by

temperature in lab studies (Fischer and Whalen 2005, Maag et al. 1997).

The limiting factor for denitrification varies among ecosystems, as well as in microsites

within an ecosystem. For instance, denitrification can take place in microsites of the soil profile

if a soil is well-drained with seasonal wetting periods. This creates anaerobic "hotspots" within

the soil profile where denitrification can take place. High carbon microsites are also

hypothesized to be a maj or source of error for rates of denitrification measured. Knowledge of

soil properties, hydrology, climate, and the biotic community can help predict how effective a

system will be at removing nitrate through denitrification. This may be especially important near

agricultural areas with connections to groundwater.










Nitrogen and Florida's Waters

Waterways impacted with nitrates are especially problematic in areas where direct

connections between ground and surface waters occur. These connections can lead to the

contamination of aquifers, and, subsequently to sources of drinking water, especially near

industrial and agricultural landscapes. In many parts of Florida, connections can be numerous as

a result of geology.

Limestone from the state's marine origins lies beneath Florida soils. A geological

formation known as karst forms when limestone comes into contact with carbonic acid. When

CO2 is dissolved in water from the break-down of organic matter, it forms carbonic acid

(H2CO3). As carbonic acid comes into contact with limestone, calcium carbonate is easily

dissolved. Over time, holes in the limestone develop from this erosion process forming karst.

Some examples of karst formations in Florida are caves, springs, and sinkholes, all of which

provide a conduit between surface and ground waters.

In most of Florida, this direct connectivity is not a concern because an impermeable layer

of silt and clay, called the Hawthomne layer, underlays the soil. The Hawthomne formation was

formed by the deposition of phosphorus-rich clay and sand from ancient rivers and can be as

deep as 800 ft in parts of western Florida. When the Hawthorne layer is intact, there is no direct

connection to the Floridan aquifer. In the north-central portion of the state, however, along the

Ocala Uplift, the Hawthomne layer has thinned so limestone is within 0-50 ft of the ground

surface (Figure 1-1). The interface zone between intact and eroded Hawthorne layer is called the

Cody Scarp. Along this interface, thinning of the Hawthorne layer allows increased infiltration

of surface water to underlying limestone leading to dissolution and occasional collapse forming

sinkholes. Once the Hawthomne layer is completely eroded, direct leaching of surface waters and

rainfall through the soils to the aquifer is possible. Interaction between surface water and










groundwater along this zone is significantly increased and can lead to water quality degradation

within the aquifer.

Agriculture management practices in areas where karst formations are present can have

significant ecological impacts on ecosystems and watersheds from applied fertilizers or animal

waste. These non-point sources are subj ect to runoff and leaching into ground and surface

waters, introducing nitrogen to waterways that otherwise might be nutrient limited. One area

that illustrates the change in hydrologic connectivity along the Cody Scarp and potential impacts

of agricultural activities due to these connections is the Santa Fe River Watershed.

The Santa Fe River Watershed

The Santa Fe River watershed covers 3,585 square kilometers in north central Florida and

drains into the 121 km long Santa Fe River. This watershed lies within the Suwannee River

Basin that drains to the Gulf of Mexico (Figure 1-2). This area of Florida typically receives a

mean annual precipitation of 1.3 meters and has a mean annual temperature of 24oC.

Dominant land use types in this area of Florida are silviculture, row crop and pasture

agriculture, and undeveloped natural areas (Figure 1-3). In the upper and middle watershed,

agricultural and timber production areas are of concern because fertilizer and animal waste may

be susceptible to runoff and leaching into tributaries, creeks, and springs. Along the Santa Fe

River, numerous tributaries drain agriculture areas that contribute water and nutrients to the river

(Figure 1-4). Major tributaries include the Ichetucknee, Olustee, New, and Sampson Rivers.

Because of the geology of this region, these surface waters can come into contact with

karst formations through sinkholes. The river actually enters a major sinkhole near the Cody

Scarp and goes completely underground for 5 km and re-emerges before entering the Gulf of

Mexico (Figure 1-4). Therefore surface waters in the upper and middle Santa Fe watershed will

eventually enter groundwater and then eventually drain to freshwater and marine systems










possibly leading to eutrophication, aquifer contamination, algal blooms, and other degradations

in water quality. Possible nitrate sources in this watershed include septic tanks, atmospheric

deposition, fertilizers, and animal waste.

Objectives and Hypotheses

The main goal of this study was to characterize water quality in several tributaries of the

middle Santa Fe River watershed and to determine the extent to which riparian soils can

effectively reduce nitrate concentrations in waters impacted by agriculture. Specific obj ectives

were to

evaluate spatial nitrate-nitrogen dynamics in tributaries and riparian wetlands at the
Boston Farm Santa Fe River Ranch Beef Unit;

identify reaches within the tributaries that may have a greater capacity to remove
mitrate;

determine if carbon or nitrogen is limiting denitrification in these riparian areas;

determine the denitrification potential in a riparian wetland zone characteristic of
tributaries in the Santa Fe River basin;

Findings from studies that addressed these obj ectives are outlined in the following

chapters of this thesis. In Chapter 2, nitrate concentrations as well as other nutrients in surface

waters of the two tributaries are discussed.

Soil characteristics and denitrification potential of soils along the tributary and adj acent wetlands

are addressed in Chapter 3. Chapter 4 is a summary chapter to discuss implications of these

finding and suggestions for future research.






































Figure 1-1. Along the Ocala Uplift in eastern Florida, the Hawthorne layer becomes
discontinuous, allowing surface and groundwater connections to occur. (Reprinted
with permission from The Florida Speological Society, Gainesville, Florida,
http ://www.caves.com/fss/pages/misc/geology .htm.)












































Figure 1-2. Location of the Santa Fe River and Suwannee River, relative to the Cody Scarp
(Reprinted with permission from Martin, J, Screaton, E., and Moore, P.2004. Surface
and ground water mixing along the Cody Scarp: An example from the Santa Fe River
Sink-Rise system. USGS Suwannee River Basin and Estuary Integrated Science
Workshop Proceedings.

























































Data Sources Suwannee Plver Management D str ct, 1 40 P00, 1995 Map projecton by Adrienne Fr~sbee





Figure 1-3. Dominant land uses common in the Santa Fe River Watershed





















































Figure 1-4. A digital elevation map of the Santa Fe River Watershed that shows the repeating
pattern of tributaries and where the river goes underground in the western portion of
the watershed










24









CHAPTER 2
WATER QUALITY MONITORING

Introduction

Numerous tributaries drain agriculture areas leading to the Santa Fe River that eventually

drains to the Gulf of Mexico. High concentrations of nitrate in water can be detrimental to

humans, and to marine or freshwater ecosystems. The research and development of methods to

decrease nitrate in water is of interest especially where groundwater will be impacted. If a

sufficient riparian buffer exists along these tributaries, there is the potential for nitrates and other

nutrients to be reduced in the water column through denitrification or plant uptake. Water

column nitrate concentrations may also decrease when freshwater systems are diluted by surface

runoff or groundwater intrusion.

Tributaries of the Santa Fe River may have spatial differences in water quality as a result

of biotic and abiotic factors. For instance, some reaches of a tributary may have plants that are

able to immobilize nitrate from the water column. Other areas may be anaerobic with a labile

carbon source, conditions ideal for denitrification. Other tributary reaches may be too

channelized or sandy for significant nitrogen removal to occur. As a result, nitrate removal

efficiencies in tributaries impacted by agriculture may vary along the length of each tributary.

Nitrate concentrations in tributaries draining agricultural areas may also have seasonal

variations in water quality. For example, irrigation and fertilization practices are maximized at

different times of the year according to plant needs, which can cause nitrate concentrations to

vary in tributaries.

Precipitation and evapotranspiration can also alter tributary nitrate concentrations by diluting or

concentrating nitrates. While long-term studies are necessary to thoroughly understand seasonal

changes, inferences can be made on what may be driving measured fluctuations over time.










In order to better understand nitrate-nitrogen dynamics in tributaries of the Santa Fe River

Watershed, a one year study was conducted on two tributaries that drain to the Santa Fe River at

the Boston Farm- Santa Fe Beef Research Unit (SFRBU).

Objectives and Hypotheses

The maj or goal of the water quality study was to understand the fate of nitrate in two

tributaries that drain to the Santa Fe River.

Specific obj ectives were to

1. make observations of how nitrate concentrations vary from month to month;

2. determine if different stream reaches remove more nitrate than others.

Specific Hypotheses:

1. Nitrate concentrations will vary over the course of the study as a result of season
changes, fertilization, or irrigation. It is expected that highest fertilization rates will
be in spring and summer, and therefore highest nitrate concentrations will occur
during these months.

2. Some reaches in the tributaries will be more effective at removing nitrate from the
water column than others. Reaches with plants in the water column or an available
carbon source are hypothesized to remove more nitrate than sandy channel reaches
without plants.

Materials and Methods

Site Description

The Boston Farm -Santa Fe River Ranch Beef Unit (SFBRU), a University of Florida

property, provides an excellent representative site of typical landuse and topography along the

middle third of the Santa Fe River. The research site is located about 30 miles northeast of

Gainesville, Florida in Alachua County (Figure 2-1).

Soils in this watershed are sandy and are predominantly Ultisols, Spodosols, and Entisols.

Specific soils in sampling areas are Sparr fine sand, Pelham, Plummer, and Masotte soils, and

Chipley sand (SSURGO). The site has a number of features characteristic of north central










Florida's geologic and biotic communities. These include groundwater seeps, sinkholes,

tributaries, ponds, and wetland communities.

Land use on the site consists of low intensity pastures bordered by forests and riparian

areas associated with the tributaries. The research unit supports a low density cattle operation

with about 300 heifers on 1,600 acres. Adj acent to the property is a plant nursery that is

potentially responsible for elevated nitrate concentrations measured in water sampled from the

site in 2004.

Two tributaries run the length of the property and drain to a floodplain leading to the Santa

Fe River (Figure 2-2). Tributary 1 (T1) drains cattle pastures on the Santa Fe Beef Research

Unit SFBRU as well as The Holly Factory, an ornamental plant nursery adj acent to the research

site. During this study, cattle were only observed in the pasture bordering this tributary during

the month of October. Tributary 2 (T2) had less flow than T1 and at times went underground or

had low water levels during the sampling period. Cattle are kept out of this tributary by barbwire

fencing, although runoff can still enter the stream from nearby pasture and from upstream during

larger rainfall events

Field Methods

To address the hypotheses posed in this study, two tributaries in the Santa Fe watershed

were selected on the Santa Fe River Beef Research Unit -Boston Farm (Figure 2-2). Along

these tributaries, we designated transitional zones between morphologically different stream

reaches. Eight morphologically discrete stream segments were designated using dominant

vegetation type, degree of bank incision, and whether depositional or erosional processes were

the principal drivers along the reach (Table 2-1, Figures 2-2 to 2-9). These classifications can be

compared to the widely used Rosgen stream classification system which uses shape, slope and










pattern to classify streams and rivers (Rosgen and Silvey 1996). The Rosgen classification,

however, does not take into account dominant vegetative community.

Once stream reaches were classified according to the above criteria, monthly water

samples were taken at the beginning and end of each reach. This sampling method led to a total

of 20 sampling stations along T1 and 10 along T2. For each sample station, an acid washed 250

mL bottle was rinsed three times with site water before collecting a sample. Care was taken to

collect samples from an undisturbed portion of the water column in the middle of the channel at

mid-water column depth. Water samples were then acidified to a pH of 2 with ultra pure

concentrated sulfuric acid and put on ice for transport to the lab. In the lab, samples were

transferred to scintillation vials. Samples to be analyzed for NO3 N4 and DOC were filtered

with a Whatman 0.45Clm filter. Samples analyzed for total Kj eldahl nitrogen (TKN) were not

filtered.

In May 2005 and March 2006, water samples were collected in T1 from the end of the

tributary, through the floodplain and improved pasture up to the Santa Fe River, this resulted in

an additional four samples. These samples were analyzed for NO3-, SRP and Cl-.

Analytical Methods

All water samples were refrigerated and analyzed for nutrients within 28 days as

recommended by the EPA. Nitrate, ammonium, and TKN were analyzed to determine the

dominant nitrogen forms present in the water column. Nitrate was analyzed colorimetrically

using the cadmium reduction method on a rapid flow or a discrete analyzer (EPA method 353.2).

Ammonium was analyzed colorimetrically on a Technicon AAIII autoanalyzer (EPA. 350.1).

TKN was determined by digesting the water samples with sulfuric acid and a copper sulfate

mixture to convert organic forms of nitrogen to ammonium. Ammonium was then analyzed

colorimetrically on a Technicon AAII (EPA. 351.2).









Dissolved organic carbon (DOC) and soluble reactive phosphorus (SRP) were analyzed

because of their potential roles in nutrient limitation to plants and microbes, which can in turn

affect the nitrogen cycle. DOC was analyzed on a Shimadzu TOC 5050a (EPA 415.1). SRP was

measured colorimetrically on a spectrophotometer (EPA 365.1).

Chloride concentrations were measured to determine if nitrate concentrations in the

tributaries were being diluted by surface runoff or groundwater intrusion. Chloride

concentrations were analyzed on a Dionex lon Chromatograph.

All above methods used the QA/QC requirements set by the Wetland Biogeochemistry

laboratory which require a spike, repeat, standard, and blank to be run for every 20 samples

analyzed.

Statistical Methods

All statistics were analyzed with JMP IN 5.1. T-tests were used to compare two means,

and ANOVAs followed by Tukey-Kramer tests were used when comparing more than one mean.

All data were tested for a normal distribution and transformed if necessary before performing

analyses.

Results

Nitrate

For eleven months of sampling, nitrate concentrations in T1 had an average nitrate

concentration of 4.73 A 1.01 mg L^1 (mean & SD). Nitrate concentrations in T2 were

significantly lower than nitrate concentrations in T1 (p< 0.001) for all months, with an average

of 0.03 & 0.03 mg L^1 (Figure 2-10).

Nitrate concentrations in T2 were consistently low and showed no significant spatial or

temporal variability. Therefore, the remainder of this chapter will focus on T1 for further









analyses of tributary nitrate-nitrogen dynamics. Nitrate concentration decreased by 13-81%

from headwaters to discharge in T1 with an average reduction of 31% (Figure 2-1 1).

The Open Water (OW), Depositional Herbaceous (DH), Moderately Incised Herbaceous

(MIH), and Slightly Incised Woody (SIW) reaches all removed significantly more nitrate per

meter than the other six reach designations (Table 2-2).

Because of the relatively short sampling period, statistics were not performed to determine

if differences existed between months or seasons. There were, however, considerable

differences in mean tributary nitrate concentration between months and seasons that may be

related to fertilization, irrigation, cattle grazing, or seasonal climate differences (Figures 2-12

and 2-13).

Ammonium and Organic Nitrogen

TKN and NH4' WeTO HOt significantly different in the two tributaries (Table 2-3). T1 had

NH4+ COncentrations of 0. 14 & 0. 11 mg L^1 and TKN concentrations of 0.51 & 0.32 mg L^1. T2

had NH4+ COncentrations of 0.22 & 0.23 mg L^1 and TKN concentrations of 0.43 & 0.29 mg L^1

Dissolved Organic Carbon and Soluble Reactive Phosphorus

T2 DOC concentrations were significantly higher than T1 concentrations (p= 0.005). T1

had an average DOC concentration of 5.79 mg L^1 + 1.59, whereas T2 had an average of 12.94

mg L^1 + 8.69.

SRP concentrations were analyzed for the March 2006 sample event. SRP concentrations

were not significantly different in the two tributaries (p= 0.5). In T1, SRP concentrations

decreased 72% along the length of the tributary (Figure 2-14). In T2, SRP was reduced by 60%,

but increased when the tributary reached the floodplain (Figure 2-15).









Chloride

Chloride concentrations measured in T1 remained similar along the tributary until the

sampling station just before the floodplain where it increases from 6.5 to 13.0 mg L^1 (Figure 2-

16). Chloride concentrations were more variable in T2, with a maximum concentration of 8.2

mg L^1 (Figure 2-17).

Floodplain

Nitrate

Nitrate in T1 was reduced another 81% in May 20, 2005 and 86% in March 29, 2006 from

the last station in T1 through the floodplain and improved pasture to the Santa Fe River (Figure

2-18).

Phosphate

March 29, 2006, SRP was reduced 10% as T1 went through the floodplain to the river

(Figure 2-19).

Discussion

T1 and T2 were not significantly different in NH4+ Or TKN concentrations. NO3

concentrations, however, were significantly higher in T1 than in T2. Because both tributaries are

bordered by cattle pastures, T1 is believed to be significantly higher in nitrate as a result of

runoff from landuse practices in the upper watershed which in Figure 2-2 can be identified as a

horticultural nursery. T1 receives irrigation and storm water from the nursery, which is fertilized

year round with NH4NO3, urea, and KNO3 (T. Stevens personal communication 2006). Nolan

and Stone (2000) sampled over 50 sites across the United States and found that the maj or source

of nitrogen to groundwater was found to be from fertilizers rather than manure or atmospheric

deposition. Average nitrate concentrations in groundwater were shown to be highest near

agriculture areas (3.4 mg L^1) when compared to urban areas (1.6 mg L^1) and maj or aquifers









(0.48mg L 1) (Nolan and Stone 2000). Although our study did not address nutrients in

groundwater, it did find T1 surface waters to be impacted certain types of agricultural runoff.

Over a year of monitoring, T1 consistently showed a reduction of nitrate in the water

column as water moved from headwaters to discharge. Many studies have shown that riparian

areas can reduce nitrates in runoff before it reaches freshwater systems (Fennessy and Cronk

1997). This study, however, shows a reduction in nitrates in the stream channel. This may be a

result of plant uptake, denitrification, or dilution by ground or surface waters. Chloride

concentrations measured in this tributary showed no maj or change along the length of T1

suggesting that the decrease in nitrate concentrations is not due to a dilution by groundwater or

surface runoff, but rather from plant uptake or denitrification.

Studies have shown phytoplankton and plants remove substantial amounts of nitrogen from

water systems (Schaede and Lewis 2006, Bledsoe et al. 2004). Phlips et al. (2002) found

phytoplankton in the Indian River Lagoon, Florida were most often limited by nitrogen.

Phytoplankton populations were frequently observed at the SFBRU in the Open Water reach and

in the floodplain (in winter), thus phytoplankton may provide a sink for nitrate in T1.

Phytoplankton has also been shown to increase rates of denitrification by providing a labile

carbon source to microbes (Sirivedhin and Gray 2006) Reaches with a closed tree canopy,

however, are likely light limited, which would inhibit phytoplankton growth. Indeed, reaches

with woody species showed little to no nitrate removal in the water column.

Smialek et al. (2006) showed higher rates of denitrification in soils with herbaceous

species (Juncus sp) when compared to soils with woody species (Salix sp) present. Both woody

and herbaceous plant species occur along the tributaries, and aquatic plant species grow in some









stream reaches. Both plant and algal species in these tributaries may have a role in the decrease

in nitrate concentrations of the water column.

The decrease in nitrate was not uniform along the length of the tributary; some reaches

removed more nitrate than others, while some reaches released nitrate into the water column.

The Open Water, Depositional Herbaceous, and Moderately Incised Herbaceous reaches

removed the most nitrate from T1 during the year of monitoring. Numerous characteristics may

contribute to high nitrate removal in the Open Water. For instance, water has a long residence

time in this reach, which increases contact time with soils, plants and phytoplankton. The long

residence time also leads to deposition and build up of organic matter. The sediments in the

stream channel are constantly flooded and likely anaerobic, making this an ideal location for

denitrification. Finally, this reach has a number of species of aquatic plants along the edges of

the tributary that may be assimilating nitrate.

The Depositional Herbaceous reach is a portion of the tributary that braids through organic

and mineral deposits. These depositional areas have built up over time, and a number of plant

species are present. As water braids through these zones, it may come into contact with plant

roots that take up nitrate. The plants can also provide a carbon source for denitrification.

The Moderately Incised Herbaceous reaches have riparian and aquatic plants present that

can take up nitrate. These reaches may also be receiving DOC as it leaches from the upland. It

was, however, unexpected that this reach would remove a significant quantity of nitrate.

Nitrate concentrations in T1 did appear to vary over the course of the year. Highest mean

concentrations were observed in October 2005, the only month that cattle were observed in the

pasture directly adjacent to the tributary. As a result, nitrate concentrations did not decrease

much along the length of the tributary. Spring was found to have the highest initial nitrate









concentrations in the water column. This likely corresponds with the higher fertilizer application

rates at the beginning of the growing season at the nursery upstream (T. Stevens, nursery owner,

personal communication 2006). The most nitrate removed along the length of T1, however,

occurred in April. This is likely the result of warming soil temperatures and plant growth, which

occur in the spring. On the other hand, the least amount of nitrate was removed along the length

of the T1 in the fall. This may correspond with the release of nitrogen that occurs as plants

senesce at the end of the growing season.

Carbon quality and quantity is important to nitrogen cycling in aquatic environments

because of its effects on denitrification, nitrification, and mineralization. Strauss and Lamberti

(2000) found that glucose and leaf leachates inhibited nitrification because heterotrophic bacteria

outcompeted the chemoautotrophic bacteria responsible for nitrification. DOC was found to be

significantly higher in T2 than T1, and this may inhibit nitrification in this tributary. On the

other hand, DOC may be providing a carbon source for denitrification in T1. Finally, if a stream

has high carbon and low nitrogen, most inputs of nitrogen will be rapidly assimilated into plant

and microbial biomass (Schlesinger 1997). All of these processes could explain in part why

nitrate concentrations were significantly lower in T2 than in T1.

Available phosphorus concentrations in the water column in T1 decreased from headwaters

to discharge. Microbial and plant uptake may both have a part in SRP removal from T1.

Phosphorus may also be adsorbing on to the surface of stream sediments.

Nitrate and SRP concentrations were both dramatically reduced in May 20, 2005 and

March 29, 2006 in the floodplain. This may be due to a combination of denitrification, dilution,

and plant uptake. Although it is unknown what process is reducing nutrient concentrations, it is










clear that the floodplain is important in removing nitrate and SRP from agriculture impacted

waters in T1.

Overall, unlike T2, T1 waters were impacted by agricultural runoff. Nitrate

concentrations, however, were reduced as T1 moved from headwaters to discharge in the

floodplain. No change in chloride concentration along T1 suggests this reduction in nitrate is

from denitrification or plant uptake rather than dilution from groundwater intrusion. SRP

concentrations were also reduced along the length of the tributary.

Some nitrate reaches were more effective at nitrate removal than others, likely due to

differences in carbon availability, retention time, and plant community. Nitrate was also

significantly reduced as T1 passed through the floodplain.













Study Site-Santa Fe Research Unit
in North Central Fbloida


II I

111


















ara~mmpm aa(a ~pm da~


Ma~p t*:qerton~b Cr drrne Fn <


D~FI Savn.u36lllslr BI+NU; i9EO, i-1I~L)I
~Pulrns-Ni ~R~r Mlhtlllmrrt: Didn;l


Legend-
Sian> FE Fne agtersa


Figure 2-1. Santa Fe River Beef Research Unit relative to Gainesville, Florida and the Santa Fe
River watershed.

































Figure 2-2. The SFBRU cattle pastures with an ornamental plant nursery south of the property
and tributaries that drain to the Santa Fe River floodplain.











Table 2-1. Summary of stream reach characteristics.
Depositional or Degree of Bank
Reach type Acronym Dominant vegetation Rosgen equivalent Figure
Erosional Incision
Depositional DW tree species specificly Carya sp., Pinus sp., depositional none D or DA 2-2


woody



Depositional
herbaceous



Slightly incised
woody



Slightly incised
herbaceous


Quercus sp., M~agnolia gI~run/loratkl



herbaceous plant species such as Saururus
cernuus, Jluncus sp., Cephalanthus
occidentalis, Hydrocotle umbellata, and
Polygonum sp.
tree species specificly Carya sp., Pinus sp.,
Quercus sp., M~agnolia gI~run/loratkl



herbaceous plant species such as Saururus
cernuus, Jluncus sp., Cephalanthus
occidentalis, Hydrocotle umbellata, and
Polygonum sp.
tree species specificly Carya sp., Pinus sp.,
Quercus sp., M~agnolia gI~run/loratkl



tree species specificly Carya sp., Pinus sp.,
Quercus sp., M~agnolia gI~run/loratkl



aquatic emergent and floating plants


DH




SIW




SIH


depositional




erosional




erosional




erosional


D or DA


none




<30cm




<30cm




<50cm


Moderately incised MIW
woody


Deeply incised
woody



Open water




Floodplain


erosional




depositional




depositional


DIW




OW


>50cm




none




none


FP tree species such as Taxonium distichum and
Nyssa sylvatica



























Figure 2-3. An example of Depositional Woody stream reach.


Figure 2-4. A Depositional Herbaceous reach.


Figure 2-5. An example of a Slightly Incised Woody reach.




















E' ,r' `
.,L
'~ '
k
t~ ~
B]
''" '' '-%-. F..
jC~ u II
;;~~~~*. ~. ,a


Figure 2-6. Slightly Incised Herbaceous


Figure 2-7. Deeply Incised Woody.


Figure 2-8. An example of an Open Water reach



























Figure 2-9. A Moderately Incised Woody reach.


Figure 2-10. The Santa Fe River floodplain.




































* .- - - .


1-
r-



E 01
.-



Z

0.01




0.001


Figure 2-11i. Log of mean nitrate concentrations of tributary 1 (T1) compared to tributary 2 (T2).
Bars represent the standard deviation. T2 was not sampled in October due to the
absence of surface water.


M T1


Month










8.00


7.0 I ,1 IISIW OW DIH FP


6.00



O D~IWMI
z~ 3.00 -
SIH MIW
2.00 -

1.00 -DH MIW SIW

0.00
0 200 400 600 800 1000

Distance (m)


Figure 2-12. Average and range of nitrate concentration in tributary 1 (T1) from headwaters to discharge for all months sampled.
Samples were collected at transitional point between classified stream reaches. The reach classifications are as follows:
DW= Depositional Woody, SIW= Slightly Incised Woody, MIW= Moderately Incised Woody, MIH= Moderately Incised
Herbaceous, SIH= Slightly Incised Herbaceous, DH= Depositional Herbaceous, OW= Open Water, DIH= Deeply Incised
Herbaceous, and FP= Floodplain.








Table 2-2. Average percent change in nitrate per meter in tributary 1 (T1) for 11 months.
Values with the same letter for significance level (SL) are not significantly different.
Reach Type Mean SD SL
% ml
Open water 0.27 0.21a
Depositional Herbaceous 0.13 0.65 ab
Moderately incised herbaceous 0.08 0.09 abc
Slightly incised herbaceous 0.04 0.10 abc
Deeply incised herbaceous 0.01 0.02 c
Floodplain 0.00 0.14c
Moderately incised woody -0.01 0.36 bc
Deeply incised woody -0.04 0.20 bc
Slightly incised herbaceous -0.04 0.42 abc


















4-I









Month


Figure 2-13. Quantiles of monthly nitrate concentrations measured in T1























- spring
--- summer
fall
- winter


'9\


400


600


1000


Distance from headwaters (m)


Figure 2-14. Nitrate concentrations by season in tributary 1 (T1). Spring = March, April, May;
summer = June, August; fall = September, October, November; and winter = January,
February.













Month Mean [NH4+] Mean [TKN]
mng L'
T1
Mar-05 0.15 (0.08) 0.32 (0.07)
Apr-0 5 0.22 (0.08)-
May-0 5 0.22 (0.24)-
Jun-05 0.20 (0.35) 0.50 (0.32)
Aug-05 0.26 (0.31)-
Feb-06 -0.86 (0.25)
Mar-06 0.06 (0.02) 0.26 (0.16)
Average 0.14 (0.11) 0.51 (0.32)

T2
Mar-05 0.25 (0.11) 0.41 (0.16)
Apr-05 0.13 (0.03)-
May-05 0.27 (0.04)-
Jun-05 0.09 (0.20) 0.30 (0.05)
Aug-05 0.05 (0.03)-
Feb-06 -0.63 (0.35)
Mar-06 0.04 (0.01) 0.22 (0.04)
Average 0.22 (0.23) 0.43 (0.29)


Table 2-3. Summary of NH4+ and TKN measured in tributary (T1) and tributary 2 (T2) with one
standard deviation in parentheses. Dashes indicate months that were not analyzed for
NH4' Or TKN.






























0 100 200 300 400 500 600 700 800 900 1000

Distance along tributary (m)



Figure 2-15. SRP concentrations in tributary 1 (T1), March 29, 2006.


"0.4

S0.3

0.2

0.1


0 50 100 150 200 250 300 350 400

Distance along tributary (m)



Figure 2-16. SRP concentrations for tributary 2 (T2), March 29, 2006.
























200


600


800


1000


Distance along tributary (m)

Figure 2-17. Chloride concentrations for May 20, 2005 along the length of tributary 1 (T1).

10

9-_-v


0 50 100 150 200 250 300 350 400 450
Distance along tributary (m)


Figure 2-18. Chloride concentrations for May 20, 2005 along the length of tributary (T2).
















5

14
E





1

0


-* May-05
-eMa r- 06


0 200 400 600 800
Distance


1000 1200 1400 1600 1800


Figure 2-19. Change in nitrate concentrations as tributary 1 (T1) flows through the floodplain
and improved pasture to the Santa Fe River.















0.18

0.16 Santa Fe
0 14 River
-Tl before


0r 0.12
0.08 Atr.Following improved
Floodplain pasture After more improved
v,0.06 -pasture and hardwood
0.04 -forest
0.02
0.0

0 200 400 600 800 1000 1200 1400 1600 1800

Distance (m)



Figure 2-20. Change in soluble reactive phosphorus (SRP) concentrations from the last tributary
1 (T1) sample station, through the floodplain and improved pasture to the Santa Fe
River.









CHAPTER 3
SOIL CHARACTERIZATION AND DENITRIFICATION

Introduction

Denitrification, a maj or removal pathway for nitrogen from ecosystems, requires low

oxygen, a labile carbon source, a nitrate source, and an active microbial community. Because

wetlands are often anaerobic with an accumulation of carbon, these ecosystems can provide ideal

conditions for denitrification.

Nitrate-nitrogen concentrations in a tributary impacted by agricultural runoff are being

reduced by some mechanism as water moves along the length of the tributary (Chapter 2). To

determine the possible role of denitrification in reducing nitrate in this system, three

studies were conducted on two tributaries at the Santa Fe Beef Research Unit (SFBRU). A soil

characterization study, a nutrient limitation study, and an intact core study were conducted on soils

of the stream, bank, and upland of tributary 1 (T1) and tributary 2 (T2).

These studies were conducted to investigate soil characteristics that have been shown to

directly or indirectly influence denitrification in soils such as carbon and nitrogen (Fischer and

Whalen 2005, Lowrance 1992, Aber et al. 1991).

Objectives

Specific obj ectives were to

1. determine if denitrification is a maj or removal pathway for nitrogen from the soils of
the SFBRU;

2. determine what nutrients, if any, are limiting denitrification in this system;

3. determine if there are differences in nitrate removal rates among upland, bank, and
stream channel soils in intact cores;

4. determine if nitrate removal rates in intact soil cores correspond to denitrification
rates measured by the soil slurry method.










Hypotheses

Specific hyptheses were that

1. denitrification is a dominant pathway for nitrogen loss under anaerobic conditions;

2. because soils are mostly sandy in this region of Florida, carbon limits denitrification;

3. the bank and upland will have higher rates of nitrate removal from the water column
than the channel due to more available soil carbon;

4. denitrification measurements using intact soil cores will be more variable than the
soil slurry method because the experiment is conducted in a less controlled
environment.

Materials and Methods

Sampling Locations

Soil sampling was conducted along three transects established on a high nitrate (T1) and a

low nitrate (T2) tributary at the SFBRU. All transects run perpendicular to the tributary and are

located at the upper (headwater), middle, and lower portion (near floodplain) of each tributary.

Five sampling stations along each transect were established at the center of the stream channel, on

either bank of the main channel and 25 meters upland from each bank sampling point (Figure 3-1).

Bank sampling stations were representative of riparian areas, and the site 25 meters from the bank

was representative of upland areas. These sample sites and transects were used for all soil

sampling events.

Soil Characterization Study

Field methods

On June 20, 2005, triplicate soil samples were randomly taken within a 1 meter radius of

each sampling location along each transect. A 7 cm diameter soil corer with sharpened metal head

was used to extract a soil sample to a depth of 5 cm, making sure to minimize compaction of the

soil. A knife was used to cut any roots and to ensure the sample obtained was flush with the end of









the soil corer. Each sample was extruded into a plastic storage bag and excess air was removed

before sealing the bag. A total of 90 samples were put on ice for transport to the lab.

In addition to soil sampling, redox potential of the soil was measured four times between

November 2005 and March 2006. Redox potential is a measure of how reduced a soil is and

therefore what dominant electron acceptor is being used by microbes during respiration. To

measure redox, platinum electrodes were set up in duplicate at sample sites along the middle

transect of each tributary. Each electrode was inserted to a depth of 5 cm, the depth at which all

soil cores were taken. An Accumeter redox probe was connected to a pH meter and a platinum

electrode to measure soil redox potential. All values were adjusted to the standard hydrogen

electrode by adding 207mV to the measured value.

Laboratory methods

Once in the lab, soils were weighed for bulk density. Soils were then processed by

homogenizing samples and removing any large live and dead plant material, roots, or rocks.

Samples were stored in sealed plastic tubs at 4oC until analysis.

Soil moisture content was measured gravimetrically by drying 10-20g of soil at 70oC for at

least 72 hours. Samples were then reweighed and soil moisture content was calculated.

Soil organic matter content was determined by the loss on ignition method. Oven dried soil

samples were ground and passed through a #60 sieve (0.25mm). Any soil remaining in the sieve

was ground with a mortar and pestle until it could pass through the sieve. Approximately 2g of

dry soil was placed in an aluminum tin, weighed, and combusted in a muffle furnace for 30

minutes at 250oC then for 3-4 hours at 550oC. Ashed samples were reweighed to determine total

loss of organic matter (Jackson 1993).









Soil pH was measured by placing 10 grams of soil and 10 grams of deionized water in a

beaker and allowing the mixture to equilibrate for 30 minutes. The soil-water solution was then

measured with a calibrated pH meter (Thomas 1996 and Hanlon 1984).

To measure water extractable carbon and nitrate, 2.5g of soil and 25mL of distilled

deionized (DDI) water were added to plastic extraction tubes. A rubber stopper was placed in each

tube and the soils were shaken on an end to end shaker at an intermediate speed for one hour.

Samples were then centrifuged for 10 minutes at 6000 rpm. Extractions were filtered with a

Whatman # 41 filter (0.45Clm). Water extractable nitrate (WEN) was analyzed by the cadmium

reduction method discussed in chapter 2. Water extractable carbon (WEC) was measured on a

Shimadzu TOC 5050A.

Denitrification potential

To measure denitrification potential of these soils, denitrification enzyme activity (DEA)

was measured within 2 weeks of soil collection. This process measures the activity of microbes,

specifically denitrifying bacteria under anaerobic conditions.

For the DEA procedure, 8-10 g of soil was weighed into a 120mL glass serum bottle.

Bottles were capped, crimped, and evacuated with N2 gaS to establish anaerobic conditions. Five

milliliters of purged H20 were added to create a soil slurry.

The acetylene block method was used because acetylene gas (C2H2) blocks the final step in

denitrification when N20 is reduced to N2. Acetylene was generated by adding water to calcium

carbide rocks which immediately produces high grade acetylene gas. Twenty milliliters of

acetylene gas were inj ected into each serum bottle. Samples were put on a shaker for 30 minutes

to ensure complete mixing of acetylene throughout the soil. Eight milliliters of DEA solution (288

mg L^1 glucose, 56 mg L^1 KNO3, and 100mg L^1 chloroamphenicol) were added to each sample

and soils were put on an end to end shaker to incubate in the dark at a constant temperature of









25oC. The volume of chloroamphenicol used in this study was selected based on experiments

conducted by Murray and Knowles (1999). Gas samples were collected every 30 minutes for

organic soils and every hour for sandy soils for up to 3 hours. Samples were stored in 4 milliliters

evacuated, crimp-top, glass, serum bottles until analysis. N20 gas samples were measured on a

Shimadzu gas chromatograph 14A with a 63Ni electron capture detector. Column temperature was

30oC, detector temperature was 240oC, and injector temperature was 120oC. The carrier gas was

Argon and 5% methane. Denitrification rates were obtained by calculating the slope of the line

obtained when gas concentrations were plotted over time.

Nutrient Limitation Study

Field methods

Using the same soil transects discussed above, in January 2006, triplicate soil samples

were taken to a depth of 5 cm in the stream channel, at the east bank, and at the east upland soil

sampling locations. Triplicate samples at each location were combined into one sample. Samples

were homogenized and stored in a sealed plastic bag on ice for transport to the lab.

Laboratory methods

Soil samples were processed and moisture content and loss on ignition determined

according to the methods described above. To determine what may be limiting denitrification

rates in this system, 8-10g of soil from each sample were added to 3 serum bottles to represent

each treatment: ambient, + nitrogen (+N), and + nitrogen + carbon (+N+C). Each serum bottle

was capped, crimped, and flushed with N2. Five milliliters of N2 purged water and 20 milliliters of

acetylene were added to each sample as described above.

For ambient samples, 8 milliliters of DDI water were added to the serum bottles, and

samples were set to incubate in the dark on an end to end shaker at 25oC. Based on previous

sampling, the ambient soils presumably had low nitrate concentrations, so nitrate consumption was










expected to occur quickly. Gas samples were taken at approximately 20 min, 40 min, 2 hours, and

4 hours.

For the +N treatment, 8mL of a 5mg L^1 nitrate solution were added to each serum bottle.

This concentration was chosen because it is similar to the average nitrate concentration of the

water sampled in T1. Samples were set to incubate in the dark on an end to end shaker at 25oC.

Gas samples were taken at 1, 2, 4, 16 and 48 hours. These sample times were selected to try to

catch the linear portion of the denitrification reaction.

For the +N+C treatment, 8 mL of 5 mg L^1 nitrate solution and 4 grams of ground litter as a

carbon source were added to each sample. Litter was collected from the sample site near the

stream channel and was composed of a mix of woody (pine and oak) and herbaceous (knotweed,

Juncus sp, and grass) species. Samples were incubated in the dark on a shaker at 25oC. Based on

the analysis of the +N gas samples, samples for the +N+C reaction were sampled at: 1.5, 3, 10, 13,

and 28 hours.

All gas samples were stored in evacuated 4mL glass serum bottles. The N20 gas

concentration of each sample was measured on a Shimadzu 14A gas chromatograph.

Intact Core Study

Field methods

To measure nitrate removal capacity of SFBRU soils, an intact core study was carried out

in April 2006. Triplicate intact soil samples were taken along three transects in the T1 tributary

(described in Chapter 3) within the stream channel, east bank, and east upland of the impacted

tributary. Soil cores were also taken in the floodplain to determine the nitrate removal rate of

floodplain soils. Each soil core was taken to a depth of 5 cm with a sharpened steel head placed on

a 35 cm long, clear polycarbonate tube. Care was taken to minimize compaction when the










apparatus was either pushed or hammered into the soil. The steel head was removed and both ends

of the tube capped for transport. All collected cores were transported upright, on ice to the lab.

Laboratory methods

Site water from T1 with an initial nitrate concentration of 6.21 mg L1 was added until each

soil core was saturated and covered with 20cm of water. All flooded cores were placed in an

aquarium filled with water to moderate ambient temperature changes and maintain a neutral

hydraulic head difference between the inside and the outside of the core The water column of

each core was mixed by continuous bubbling with ambient air pumped through tubes fixed with a

1.5 gauge hypodermic needle. Bubbling rate was sufficient to keep the water column mixed and

under aerobic conditions, but not to the level that sediments became suspended. Black plastic was

placed over the entire experiment to minimize light and, therefore, deter algal growth in the cores.

Water samples and temperature readings were collected from the water column 14 times

over 8 days (time sampled = 0, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, and 192 hours).

Samples were analyzed with the Cadmium reduction method on an AQ2, a discrete

autoanalyzer, to measure nitrate concentrations in the water over time.

Following completion of the experiment, soils from the intact cores were analyzed for

organic matter, moisture content, and denitrification enzyme activity rate (DEA).

Statistics

All statistical analyses were performed in JMP IN 5.1, Sigma Plot 8.0, or Statistica. To test

differences between tributaries, a t-test was performed. To compare differences between

denitrification rates by stream location, when comparing more than two means, ANOVAs

followed by a Tukey-Kramer test were used. ANOVAs and Tukey-Kramer tests were also used

to compare differences between treatments in the nutrient limitation study.









DEA rates were correlated with soil characteristics to see what factors if any had an affect on

denitrification. Correlations were also analyzed for soil properties and denitrification rates for the

treatments. All Pearson product moment correlations were performed in JMP IN 5.1 and

Stati sti ca.

A factor analysis was performed by the Principle Component extraction method to get an

overview of how soil characteristics affect variation in soils samples by location. Factor analyses

were run with Statistica.

Differences between soil cores were analyzed with ANOVAs followed by a Tukey-Kramer

test in JMP 5.1. Correlations were run in JMP 5.1.

Results

Soil Characterization Study

Soil bulk density, pH, % moisture content, % organic matter, water extractable carbon

(WEC), and water extractable nitrate (WEN) measurements were used for initial soil

characterization (Table 3-1). When combining sites along each tributary, bulk density was

significantly higher in T1 than in T2 (p= 0.04). WEN and WEC were not significantly different

(p= 0.13 and 0.10, respectively). Finally, % organic matter, % moisture content, and pH were not

significantly different for T1 compared to T2 (p= 0.22, 0.57, and 0.12, respectively).

Each tributary had a number of differences in soil properties between upland, bank and

stream soils. Differences between locations were observed for all soil properties except pH in T1

and moisture in T2 (Table 3-1).

Overall, the mean DEA rate was 5.89 & 9.83 mg N20 kg soil-l d- T1 had an average DEA

rate of 8.73 A 12.78 mg N20 kg soil-l d which was significantly higher than T2, with an average

DEA rate of 2.50 & 2.68 mg N20 kg soil-l d-l (p= 0.04).









For both tributaries, the upland and bank soils had significantly higher DEA rates than the stream

channel soils (p<0.001; Figure 3-2). There were no differences in DEA rates between transects on

either tributary.

Percent organic matter had the strongest correlation with T1 and T2 DEA rates. DEA rate

was also correlated with bulk density and WEC in both tributaries. There was not a strong

relationship in either tributary between DEA rate and pH, WEN, or % moisture content (Table

3-2).

A factor analysis was performed by the Principle component extraction method to get an

overview of how these soil characteristics affected variation in soils samples by location (Figure

3-3). Factor 1 describes 49% of the variability in soil properties, and the parameters selected were

% organic matter, DOC, DEA rate, and bulk density. Percent organic matter, DOC, and DEA

varied together, whereas bulk density was inversely related to these soil properties. Factor 2

describes 24% of the variability in soil characteristics, and the parameters selected by the Factor

analysis were moisture content and soil NO3-. These parameters were inversely related.

Upland soils were most strongly influenced by organic matter and soil nitrate

concentration. Bank soils overlapped with all soil characteristics, but clusters existed near soil

nitrate and moisture content. Finally, stream soils were inversely related to DOC, and organic

matter, but positively related to bulk density (Figure 3-3).

Redox potentials were not significantly different between T1 and T2 (p= 0.21, n= 53). For

both tributaries, however, stream redox potentials were significantly lower than those measured at

the bank and upland (p<0.001, Figure 3-4).









Nutrient Limitation Study

Denitrification rates in the ambient and +N samples were significantly lower than the +N+C

treatment (p= 0.013). Mean denitrification rates were 0.54 & 0.64 mg kg soil- dl for ambient soils,

1.56 & 2.68 mg kg soil- d lfor + N soils, and 7.17 mg kg soil- dl for +N+C (Figure 3-5).

In this experiment, there were significant relationships between denitrification rate and

organic matter, moisture content, WEC, and WEN (Table 3-3).

When soil samples were compared by location or transect there were no differences in

denitrification rates among treatments. Denitrification rates were compared between tributaries,

however, and rates in T2 were significantly higher than T1 for all treatments (p<0.001; Table 3-4).

T1 had an average denitrification rate of 1.51 mg kg soil- d-l + 3.64 whereas T2 had an average of

5.18 & 9.18 mg kg soil- d-l

Intact Core Study

For all 30 cores, average NO3~ TemOVed from the water column was 0.67 & 0.40 mg L- d .

There were no significant differences in nitrate removed per day between the floodplain, upland,

bank or stream channel (Figure 3-6). One set of cores from the stream channel in the middle

transect were outliers and had high rates of denitrification likely due to the presence of worms.

Worms can affect denitrification either via gut denitrification or increased sediment water mixing.

When cores were analyzed without these soils, the channel soils had significantly lower rates of

denitrification than the bank soils (p=0.04, Table 3-5, Figure 3-6).

There were also differences in nitrate removal rate by transect. The middle transect had an

average NO3~ TemOVal rate of 1.02 & 0.42 mg L-1 dl and was significantly different from the upper

transect (0.49 & 0.39 mg L- d- ; p= 0.19) but not the lower transect (0.66 & 0.10 mg L- d- ). The

lower and upper transects were not significantly different.









Nitrate removal rate in the core water column was significantly correlated with DEA rate,

moisture content and organic matter, but not with other soil parameters (Table 3-6).

The soil cores had an average DEA rate of 29.62 & 42.84 mg kg soil-l d- however, this

rate is much higher because it includes the floodplain DEA. Without floodplain rates, the mean

DEA rate was 16.49 & 17.94 mg kg soil-l d- DEA rates for the core soils were highly correlated

with organic matter and moisture (Figure 3-7 and 3-8). DEA was also significantly correlated with

WEC (r = 0.57), but not WEN (r= 0).

Discussion

Soil Characterization Study

For all soil characteristics measured in T1 and T2, only bulk density and denitrifieation

enzyme activity (DEA) rates were significantly different. DEA rates were quite variable in both

T1 and T2, but this is likely the result of microsites with high carbon or high moisture (Parkin

1987 and Tiedje et al. 1984).

DEA rates measured in this system were an order of magnitude lower than those measured

by White and Reddy in the Everglades, Florida (2003). Everglades soils, however, are peat soils

that accumulate carbon and receive waters high in nitrogen and phosphorus. Lowrance (1992)

measured a mean DEA rate of 0. 191 mg kg soil- dl on soils in the Gulf Atlantic coastal plain in

Georgia, compared to a mean of 5.89 mg kg soil ldl found in the SFBRU soils.

Because DEA is a measure of denitrification potential, the results suggest that under ideal

conditions, higher rates of denitrification will occur in T1 compared to T2. This is likely because

T1 soils have a steady nitrate source to utilize in the water column, whereas T2 has low nitrate

concentrations and thus, denitrification is limited by nitrate.

Redox potentials measured in the stream channel also show T1 redox to be in the optimum

range for nitrate reduction, whereas in T2, redox potentials are too low for nitrate to be the









dominant electron acceptor. Rates of denitrification in T1 suggest the reduction of nitrate

observed along the length of the tributary is likely in part due to denitrification.

DEA rates were significantly higher in the bank and upland soils of both tributaries

compared to soils in the stream channel. Because denitrification rates were highly correlated with

organic matter content and water extractable carbon, it is likely that the observed differences in

denitrification by stream location are related to carbon availability. T1 upland and bank soils were

significantly higher in carbon than stream channel soils and T2 upland soils were higher in carbon

than bank and stream channel soils.

Although denitrification potentials were higher in the upland and bank soils, redox

measurements show these soils were using 02 aS the dominant electron acceptor and were,

therefore, aerobic. These zones would be ideal for denitrification, but only when flooded will the

soils become anaerobic enough to carry out denitrification.

Nutrient Limitation Study

The nutrient limitation study showed that denitrification rates were limited by both carbon

and nitrogen, but most strongly by carbon. Nitrogen and carbon, however, might be co-limiting.

Fischer and Whalen (2005) measured the effect of the addition of nitrate, glucose, and nitrate +

glucose on DEA rates. Highest rates were obtained in the nitrate + glucose treatment, similar to

our findings. In their experiment, however, there were no significant differences between the

nitrate and glucose treatments.

Unlike the DEA experiments, soils in T2 had significantly higher denitrification rates than in

Tl. This is likely due to the presence of chloroamphenicol in the DEA solution, which blocks the

microbial production of new enzymes for denitrification, allowing only enzymes already present in

the soil to be used for denitrification. T2 has low nitrate availability so it is less likely that

microbes are using NO3~ aS an electron acceptor for respiration. Depending on how reduced these









soils are, microbes would be using other electron acceptors such as 02, Fe3+ Or CH4 during

respiration. The nutrient limitation study, however, does not use chloroamphenicol, so microbes

can produce new enzymes to carry out denitrification. There are likely differences between the

tributaries in microbial activity, micronutrients, or soil texture that are driving differences in

denitrification rates with and without chloroamphenicol. Studies have shown that soil texture can

affect denitrification (Godde and Conrad 2000; Groffman and Tiejde 1991). D'Haene et al. (2003)

found highest denitrification rates in clay soils (low bulk densities) and lowest rates in sandy soils

(high bulk densities). This may help explain differences in our findings for the nutrient limitation

study since higher rates of denitrification were found in T2 soils with lower bulk densities than

those in T1.

Intact Core Study

Intact soil core nitrate removal rates were highly variable, ranging from 0.01 to 1.94 mg L-

Id- When soils were initially compared by sample location, no significant differences were

found. Tubificid worms were observed in a set of cores from the stream channel of the middle

transect. Some tubificid worms are able to tolerate low oxygen conditions and often occur in low

nutrient conditions (Howmiller 1975). Although cores with worms had extremely low organic

matter content, they had the highest net nitrate removed from the water column during the

experiment. Studies have shown that tubificid worms significantly increase rates of microbial

processes because bioturbation allows surface particles and chemical species to infiltrate to lower

depths in the soil (Mermillod-Blondin et al. 2004). This could have increased the NO3- transport

rate from the aerobic water column to anaerobic sites in the soil where denitrification takes place.

When these cores were excluded from analysis, mean nitrate removal rates from channel

cores were lower than upland soils and significantly lower than bank soils. This is similar to

findings in previous experiments, and is likely due to carbon availability.









Intact core nitrate removal rates can be quite different from denitrification potentials

measured in the lab. The presence of plants and bioturbators can influence the process of

denitrification. Studies have shown that the presence of plant roots can increase denitrification

rates because nitrification can take place in the oxygenated zone surrounding roots (Hernandez and

Mitsch 2006). Nitrate is then available to diffuse to surrounding anaerobic zones in saturated soils.

This experiment did not remove small plants or plant roots from soil cores due to the soil

disturbance it would have caused in the intact cores. All cores with plants present removed more

nitrate than those without plants, but it was unclear if this was a result of plant uptake or

denitrification. In the future, it would be interesting to compare N20 emission from cores with and

without plant roots.

The DEA in intact core soils were higher than in previous experiments. These findings are

likely the result of soils in the intact cores being saturated throughout the experiment, allowing

them to become anaerobic for a longer period of time. Under anaerobic conditions, more enzymes

would be produced by denitrifying bacteria to carry out denitrification in the presence of a nitrate

source. The microbial community would be utilizing nitrate as an electron acceptor in the process

of denitrification, and nitrate concentrations in the water column would decrease.

DEA rates were also highly correlated with porosity in these soils. This may be because

larger porosities allow more nitrate to diffuse into the anaerobic portion of the soil profile.

In summary, DEA rates were higher in T1 than in T2 for the soil characterization study

likely due to the lack of denitrification occurring in the low nitrate T2. Denitrification rates were

highest in upland and bank soils compared to stream channel soils, likely due to carbon

availability. The nutrient limitation study showed that denitrification in both tributaries was

limited by nitrate and carbon. T2 denitrification rates were higher when soils were incubated for










longer time periods in the absence of chloroamphenicol possibly due to differences in microbial

activity and soils texture. Finally, in the intact core study, intact soil core nitrate removal rates

were lowest in the stream channel in the absence of tubifieid worms.

Highest nitrate removal rates were found in bank soils indicating that, when flooded, these

zones would be optimal for denitrifieation. DEA rates were also higher in these soils than in

previous experiments, likely due to the fact that soils were flooded previous to denitrifieation

potential measurements.












Upper transect


Middle transect


Lower transect


Figure 3-1. Sampling sites on each tributary. Each transect had sample stations at the upland
(U), bank (B), and stream channel (S).









Table 3-1. Soil characteristics in tributary 1 (T1) and tributary 2 (T2) for the upland, bank, and stream. Values (n=15) represent mean
and + one standard deviation. Values with different letters indicate that upland, bank, and stream characteristics are
significantly different within each tributary (oc=0.05).
Tributary 1
BD (g cm 3) pH WEN (mg kg ) WEC (mg kg~ ) %OM %Moisture
Mean 1.23 (0.32) 5.82 (0.98) 1.25 (2.45) 9.33 (5.03) 5.21 (3.67) 18.85 (12.16)
Upland mean 1.05 (0.19) a 5.43 (1.14) a 2.70 (3.41) a 10.62 (3.41) a 7.22 (1.52) a 8.36 (2.72) a
Bank mean 1.24 (0.35) a 6.00 (0.75) a 0.33 (0.40) b 10.54 (6.06) a 5.19 (4.41) a 27.50 (12.96) b
Stream mean 1.58 (0.16) b 6.22 (0.87) a 0.22 (0.33) b 4.32 (1.14) b 1.23 (0.93) b 22.56 (3.09) b

Tributary 2
BD (g cm 3) pH WEN (mg kg ) WEC (mg kg~ ) %OM %Moisture
Mean 1.08 (0.34) 5.55 (0.64) 2.45 (4.20) 7.76 (4.78) 6.43 (5.82) 17.30 (12.35)
Upland mean 0.89 (0.24) a 5.13 (0.54) a 4.05 (4.79) a 11.16 (5.21) a 10.17 (6.26) a 18.70 (16.41) a
Bank mean 1.06 (0.19) a 5.59 (0.40) b 0.97 (0.74) b 6.38 (2.98) b 4.91 (3.14) b 12.94 (8.49) a
Stream mean 1.58 (0.16) b 6.31 (0.50) c 0.27 (0.28) b 3.72 (0.66) b 1.00 (0.66) b 20.29 (2.27) a












p< 0.001
25


S20

r A T1
E T2

10


a 5-



io Upland Bank Stream


Figure 3-2. Mean denitrification enzyme activity (DEA) rates in tributary 1 (T1) and tributary 2 (T2) in the upland, bank, and stream.
Error bars represent one standard deviation.










Table 3-2. Pearson product moment correlations (r value) between denitrification enzyme activity (DEA) rates and soil characteristics
for tributary 1 and 2.
Soil Parameters
Tributary Bulk Density pH WEN WEC %OM %Moisture
1 DEA rate 0.73 0.35 0.33 0.69 0.80 0.08
2 DEA rate 0.60 0.07 0.32 0.50 0.70 0.12





















1


0


LI.


OO O


n


Soil
I nitrate


II

BD i


Q
Organic matter


I e W

DEA rate


EC


Moisture
content


SBank
A Stream
O Upland


Factor 1 = 49% of variation


Figure 3-3. Factor analysis of soil characteristics and stream location.





PA


600

400

200

0

-200


mT1
m T2


1400 '
Upland Bank Stream
Location



Figure 3-4. Mean redox potentials in tributary 1 (T1) and tributary 2 (T2). Nitrate is the
dominant electron acceptor for redox potentials from 200-250mV. Error bars
represent one standard deviation.


p= 0.013


N and C added


Ambient


N added


Treatm ent


Figure 3-5. Mean + one standard deviation of denitrification rates for each treatment of a
nutrient limitation experiment (N= nitrogen, N and C= nitrogen and carbon).









Table 3-3. Pearson product moment correlations (r value) between denitrification rates of each
treatment and soil characteristics.
Soil parameter Ambient N added C and N added
% Organic matter 0.50 0.73 0.66
% moisture 0.26 0.39 0.16
WEC 0.40 0.74 0.55
WEN 0.24 0.56 0.43


Table 3-4. Mean & one standard deviation of denitrification rates for each treatment in tributary
1 (T1) and tributary 2 (T2).
Treatment Tl T2

mng kig soil d'
Ambient 0. 19 & 0. 13 0.73 & 0.71

(+) N 0.38 & 0.33 2.74 & 3.46
(+) N (+) C 3.18 & 5.34 1 1.17 & 13.28


2-
1.8-
1.6 -
1.4 -
1.2-




0-


SLowrer Middle Upper
transect transectt transect


3~`t-


~
~


,~t- a~


~ ~


Location


Figure 3-6. Mean nitrate removal rates + one standard deviation of each set of intact soil cores.
The upland of the upper transect was not sampled because of equipment problems.
Tubificid worms were found in the set of three cores taken from the channel of the
middle transect.







Table 3-5. Mean nitrate removal rate + one standard deviation by sampling site. Mean values
followed by the same value are not significantly different. This analysis excludes a
set of cores that were outliers.
Location Nitrate removal rate SD

mg L'd'
Upland 0.67 ab 0.26
Bank 0.79 a 0.14
Stream 0.44 b 0.33

Table 3-6. Pearson product moment correlations between nitrate removal rate per day and soil
characteristics (DEA is denitrification enzyme activity, WEC and WEN are water
extractable carbon and water extractable nitrogen, respectively).
Soil Parameter r

Organic matter (%) 0.3 8
Moisture content (%) 0.36
DEA rate (mg kg' d ) 0.47

WEC (mg kg soil ') 0.22

WEN (mg kg soil ') 0.16

















y = 1.33x + 0.53
R2 = 0.677




t*


7 00.5 1.0 1.5 2.0 2.5 3.0 3.


Organic Matter (%/)


Figure 3-7. Linear relationship between
(DEA) for the core soils.


organic matter and denitrification enzyme activity


200


150


100
-


LIJo


y =324. 9x -84. 3
R2 =0.868









15 0.25 0. 35 0.45 0.55 0.65 0.


Porosity


Figure 3-8. Linear relationship between porosity and denitrification enzyme activity (DEA) rate
for core soils.









CHAPTER 4
SUMMARY, IMPLICATIONS AND FUTURE RESEARCH

Water Quality Monitoring

Many tributaries of the Santa Fe River drain agricultural land in north-central Florida.

There are direct connections between ground and surface waters in this region as a result of a

discontinuous clay layer (the Hawthorne layer) that overlays limestone bedrock. When water

runs off agricultural areas, waters high in nitrogen, phosphorus, and other nutrients can impact

fresh and marine waters. Nitrate-nitrogen can affect the health of humans, animals, and

ecosystems, so improvements in management of agricultural runoff are important to maintain

Florida's drinking water quality and ecosystem health.

Two tributaries of the Santa Fe River were studied for one year. One tributary had

consistently low nitrate concentrations, while the other was high in nitrate from runoff of an

ornamental plant nursery. Our study found that nitrate is being at least partially reduced in the

impacted tributary as a result of denitrification processes prior to water reaching the Santa Fe

River. Nitrate concentrations varied over the year of monitoring likely due to season, fertilizer

application rates, and irrigation rates.

Our results indicate that open water reaches and reaches with herbaceous vegetation of this

tributary are more efficient at nitrate removal than others. To decrease nitrogen loading to

Florida's waters, a number of Best Management Practices (BMPs) could be implemented that

create or enhance these open water and herbaceous vegetation reaches in tributaries of the Santa

Fe River. For instance, small dams or weirs could be installed to decrease flow or pool water to

create open water reaches. To create reaches with herbaceous vegetation, organic and mineral

material could be deposited in a tributary. After stabilization, these depositional areas could be

planted with native hydrophilic herbaceous plants.










Soil Characterization and Denitrification

Our denitrification experiments showed that bank and upland soils had the highest

denitrification potentials. Unless the stream channel overflows, however, these aerobic soils are

not active sites for nitrate removal from the water column. BMPs could be implemented that

decrease bank incision to encourage water to overflow banks or increase contact with riparian

and upland soils. Otherwise, greatest denitrification will only occur in these soils during storm

events .

To increase denitrification occurring in the stream channel, where our findings suggest

carbon is limiting, adding a carbon substrate of some sort may be feasible. One such BMP

employs a "denitrification wall" that is constructed in water systems to improve nitrate removal.

These walls have been shown to greatly improve nitrate removal capacity by providing a carbon

source for the process of denitrification (Greenan et al 2006). These walls are long-lasting,

inexpensive, and easy to install. Common carbon sources are sawdust, peanut shells, wood chips

and plant residues. In fact, a denitrification wall is planned for installation upstream of T1 in

conjunction with the plant nursery. Schipper and Vojvodic-Vukovic (2001) found that after five

years, a denitrification wall had the same performance, and only when the water table dropped

below the wall did nitrate concentrations downstream increase.

The nutrient limitation study indicated that nitrate and carbon are both limiting

denitrification in these soils. The intact core study also indicated that denitrification is limited by

carbon, and low oxygen, anaerobic conditions. Saturation of upland and bank soils could

significantly increase denitrification in this system and improve nitrate removal from tributaries

in the Santa Fe River. Intact soil core nitrate removal rates were more variable than DEA rates

measured in the soils likely because intact soil cores are more representative of field conditions.









Future Research

This research focused on two tributaries in the Santa Fe River Watershed. A study that

monitors multiple tributaries across the watershed would be helpful to address if processes across

tributaries are similar. A number of BMPs could then be implemented and tested for success to

decrease the impact that agricultural areas have on the Santa Fe River and other freshwater

systems in the Santa Fe River watershed.

More research is also needed to understand what role plants have in removing nitrate from

reaches of these tributaries. Plants do not provide a long-term sink for nitrogen and, thus, if they

are removing nitrogen from the system, it may be helpful to harvest the plants to prevent the

release of stored nitrogen back into the tributaries.

Soil samples from different tributary reaches could also be tested for denitrification

potentials. This would increase understanding of the role of denitrification in individual reaches

within the stream channel. To increase nitrate removal, construction of similar reaches along

tributaries, or increasing the residence time of water in these tributaries could greatly increase

nitrate removal efficiencies. For instance, in this study, Open Water and Depositional

Herbaceous reaches removed the most nitrate from the water column. Open Water reaches can

be created in a tributary by narrowing the tributary channel downstream from a reach.

Depositional Herbaceous reaches can be constructed by depositing materials within the stream

that will recruit plants and force a shallow water column to flow over the reach.






The floodplain was shown to reduce both nitrate and SRP concentrations in T1, and

floodplain soils had high DEA rates. More research would be useful to study the importance of










the floodplain in reducing nutrients in agriculture runoff. It may be beneficial to restore

abandoned pastures and other land to floodplain to reduce N and P loading to the Santa Fe River.

Conclusion

This study provided evidence that a tributary of the Santa Fe River reduces nitrate

concentrations in agricultural runoff. Denitrification is believed to be a maj or process reducing

nitrate concentrations, though a combination of carbon, nitrogen, and saturated anaerobic soils

are limiting denitrification. BMPs such as denitrification walls and morphological stream reach

enhancements are suggested to increase nitrate removal from tributaries near agriculture areas.

These BMPs, however, could alter stream ecosystem function, so, it would be ideal to manage

agriculture runoff before it enters water systems. This involves reducing fertilizer applications,

intercepting runoff with buffer strips, or controlling on-site drainage (Hey 2002). Reducing or

optimizing fertilizer applications would also decrease greenhouse gases produced as by-products

of denitrification. There is currently little incentive for agriculture, industry or municipalities to

regulate nitrates.









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BIOGRAPHICAL SKETCH

Adrienne Elizabeth Frisbee was born September 23, 1979, in Dallas, Texas. She grew up

in Tulsa, Oklahoma and discovered her love of science early in biology class at Bishop Kelley

High School. When she graduated in 1998, she moved to New Orleans, Louisiana to attend

Loyola University. There, she majored in biology with a minor in environmental studies. She

graduated cum laude in 2002.

For the next two years, Adrienne was a biologist in New York, California, and Oklahoma

studying vegetation restoration and coastal and grassland bird species. After deciding to

continue her education, she moved to Florida in 2004 to get her Masters of Science degree in soil

and water science. There she studied wetlands and water quality and was also involved in

research proj ects in Alaska.

After graduating in May 2007, Adrienne will be working in San Francisco, CA with

NASA on nitrogen cycling in microbial mats.