WIIHHHIININIWIll!iii}llii‘i‘iilHllllHfllill1”HIM A-L 400 O l-I-H I FHESiS \ hi1?) Illllllllllllllllllllllllllllllllllll 31293 O 575 5717 LIBRARY Michigan State University This is to certify that the thesis entitled ASSESSMENT OF THE ORIGIN AND FATE OF NITRATE FROM SOIL .LYSIMETERS USING STABLE NITROGEN ISOTOPES presented by .Keith E. Knoke has been accepted towards fulfillment of the requirements for M.S. degree in Geological Sciences MW F621» Major professor Date ll/Sv/‘i 7‘ 0.7639 . . . MS U is an Affirmative Action/Equal Opportunity Institution —-v———’_."_~.'. W“ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MTE DUE MTE DUE DATE DUE 11” W14 ASSESSMENT OF THE ORIGIN AND FATE OF NITRATE FROM SOIL LYSIMETERS USING STABLE NITROGEN ISOTOPES By Keith Edwin Knoke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Geological Sciences 1997 ABSTRACT ASSESSMENT OF THE ORIGIN AND FATE OF NITRATE FROM SOIL LYSIMETERS USING STABLE NITROGEN ISOTOPES By Keith Edwin Knoke The concentration and 615N of nitrate in groundwater samples collected from two mid-Michigan lysimeters beneath farmland were determined on a weekly basis from March to November of 1993. The soil within one of the lysimeters was tilled and the other was not tilled. Nitrate concentrations in the samples from the tilled lysimeter were approximately twice those of the untilled lysimeter throughout the study. The nitrogen stable isotopic abundances varied similady on a seasonal basis in the tilled and untilled lysimeters. The 615N values ranged between -3.9 0/00 in the spring to 9.4 0/00 in fall (September). An increasing trend in the 615N of nitrate from both lysimeters occurred from spring to late summer, that was likely the result of fractionation due to denitrification and mineralization of the less readily mineralizable organic fraction. A decrease in 615N was observed from the middle of September to early November that reflected a decrease in the influence of microbial denitrification on the isotopic composition of nitrate. \ It." ACKNOWLEDGMENTS I would like to thank Dr. Nathaniel Ostrom for all his help and patience during the time in which it took to complete this project. I would also like to thank my thesis committee members Dr. Grahame Larson, Dr. Lars Hedin and Dr. Steve Hamilton for valuable and thought provoking comments. Partial funding for this project was made available through the Long Term Ecological Research (LTER) program at KBS (NSF DEB 92-11771). The sample collection and analysis of N03 'concentrations was completed by Mike Brown, Jeff Hamelink, and Brian Kennedy of KBS. Without their help, this project would have been difficult to complete. In addition, I would like to thank fellow graduate students Jon Kolak, Michelle Thompson and Alan McCloud for providing the needed encouragement and just as needed distractions. Last and most importantly I would like to thank my wife Amy, who, in her caring way kept reminding me and encouraging me to complete this project. TABLE OF CONTENTS UST OF TABLES ............................................................................................... vi LIST OF FIGURES ............................................................................................. vii CHAPTER 1: INTRODUCTION ........................................................................ 1 Stable IsotopeTechnique .................................................................. 2 The Microbial Nitrogen Cycle .......................................................... 4 Source of Inorganic Nitrogen to Groundwater ......................... 7 The 615N of Nitr te Sources to Groundwater ........................... 8 Isotopic Fractionation in the Microbial Nitrogen Cycle ...... 11 Isotopic Composition of Soil Nitrogen ........................................ 12 Isotopic Composition of Precipitation ....................................... 13 Field Investigations ............................................................................ 1 3 Synopsis .................................................................................................... 14 CHAPTER 2: MATERIALS AND METHODS ................................................... 1 6 LysimeterHistories ............................................................................ 1 6 Sample Collection and Preparation .............................................. 17 IsotopicAnalysis .................................................................................. 1 9 Correction for Background ................................................................ 27 CHAPTER 3: RESULTS AND DISCUSSION .................................................. 33 Results ................................ -- - ................. - ..... --_- 33 615N of Precipitation and Soil Organic Nitrogen .................... 38 1V Linear Regression Analysis of Factors Controlling the 615N of Groundwater Nitrate ........................................ Discussion ............................................................................................ APPENDIX A: FLOW RATE DATA FOR LYSIMETERS 2 AND 9 APPENDIX B: NITRATE CONCENTRATIONS FOUND IN LEACHATE COLLECTED FROM LYSIMETERS 2 AND 9 APPENDIX C: NITRATE FLUX DATA FROM LYSIMETERS 2 AND 9 APPENDIX D: 615N OF DISSOLVED NITRATE APPENDIX E: MONTHLY RUNNING WEIGHTED AVERAGE OF 615N UST OF REFERENCES ..................................................................................... ...38 ....41 49 SO 51 52 53 ...54 Table LIST OF TABLES Chronological Review of Lysimeters 2 & 9 Crop Rotation and Chemical Application .............................................. 17 615N value for IAEA N3 and KNO3 Standards Determined Using the Sealed Tube Combustion Technique ........................ 27 Percent Recovery of 20 umol of IAEA KN03 and NH4 Standards in 100 ml Deionized Water ......................................... 28 Abundance of Background NH4 Associated with the DistillationTechnique ........................................................................ 29 Abundance of Background N03 Associated with the DistillationTechnique ........................................................................ 29 615N ofNitrate Blanks ........................................................................ 31 615N Correction for Background U.S.G.S. KN03 Standard (Standard d15N = 180 0/00 ...... _ 32 615N Data for Nitrate in Precipitation ........................................ 38 .615N Data for Soil Organic Matter ................................................. 39 10. Linear Regression Analysis R2 and F-Test(F)Data ..................................................................................... 4O LIST OF FIGURES Figure 1. Microbial Nitrogen Cycle ................................................................... 6 2. Average Recovery During Distillation vs. Time: 20 umols of NH4 in 100ml & 500ml of Deionized Water ............ 21 3. Average Recovery During Distillation vs. Time: 20 umols of N03 in 100ml & 500ml of Deionized Water ............ 22 4. Changes in the 615N of the Distilled NH4 over the Course of the Distillation ................................................................. 2 4 5. Recovery of NH4 Using Zeolite with Varying pH: 20 umols NH4 in 300ml Deionized Water and 60 mg Zeolite ................................................................... -- -25 6. Percent NH4 Bounded Using Zeolite with Varying Time: 20 umols NH4 in 300ml Deionized Water and60ngeolite .................................................................................. 26 7. Seasonal Variations in the Flow Rates of LysimetersZand9 .............................................................................. 34 8. Lysimeter 2 and 9 Dissolved Nitrate Concentrations .......... 35 9. Seasonal Variation in the Nitrate Flux of LysimeterSZand9 .............................................................................. 36 10. Seasonal Variations in the 615N of Dissolved Nitrate ......... 37 11. Monthly Running Weighted Average of 615N of LysimeterZ and9 ................................................................................. 42 vii CHAPTER I : INTRODUCTION In the midwestern United States and numerous other locations around the world, nitrate contamination of ground water has become a major concern (Follett, 1989). High nitrate concentrations in groundwater have been linked to methemoglobinemia which is also know as "blue baby" syndrome (Schuphan, 1974). In response to this, the EPA has set recommended allowable limits of nitrate in drinking water of 10 mg NO3-N/L (US. EPA, 1991). The health hazard associated with elevated concentration of nitrate in drinking water is, responsible for the scientific research dedicated to the better understanding of the causes and solutions to this problem. One area of scientific research used to assess this problem is the nitrogen stable isotope technique. The nitrogen Stable isotope technique has primarily been used in determining the source of nitrate in groundwater (Kreitler, 1979, Gormly and Spaulding, 1979, Krietler and Browning, 1983, Heaton, 1984 and Flipse and Bonner, 1985). In addition to determining the source of the nitrate, the nitrogen stable isotope technique has been used to provide insight into microbial processes which may be altering the isotopic composition of inorganic nitrogen (Delwiche and Steyn, 1970, Vogel et. al., 1981, Heaton 1984, and Mariotti et al., 1988). The purpose of this thesis is to present a detailed consideration of how processes within the microbial nitrogen cycle in agricultural soils vary seasonally and as a function of tilling practices. This thesis accomplished this 1 2 objective by using the stable nitrogen isotopic technique to determine both the source of inorganic nitrogen and the microbial processes that alter its isotopic composition. STABLE ISOTOPE TECHNIQUE Stable isotope ratios are expressed in per mil ( 0/00) notation: 615N(°/00) = [(Rsample /Rstandard) - 1] * 1000 Where R is the abundance ratio of the heavy to light isotope. The internationally recognized standard for nitrogen is atmospheric nitrogen gas, which, by definition has a per mil value of O. In order to use stable isotope abundances as indicators of origins, pathways and fate of inorganic nitrogen in groundwater, two criteria must be satisfied. First, the primary sources of interest must be isotopically distinct from each other. Second, the isotopic Signature of the source must not be altered during transport and transformation (Macko and Ostrom 1994). When isotopic alteration does occur, it must be altered in a predictable manner if the origin of the nitrate is to be determined. Nonetheless, the extent of isotopic alteration can be indicative of the fractionating process. Considerable research has been conducted to improve the ability of the natural abundance stable isotope technique to quantify the extent of fractionation during equilibrium or kinetic reactions. Fractionation that occurs during an equilibrium reaction is a function of bond energies and may be calculated empirically (Urey, 1947). Fractionation that occurs during kinetic reactions is a function of the difference in mass of the isotopes. The fractionation 3 factor (a) was developed as an indicator of the extent of fractionation that may occur during kinetic and equilibrium reactions (O'Neal, 1981). In this study the reactions of interest are primarily unidirectional and kinetic. The fractionation factor for kinetic reactions: A--->B is a function of reaction rates for the light (k1) and heavy (k2) isotopes. aA-B = k] / k2 Isotopic fractionation may also be expressed in terms of the isotopic enrichment factor, at e-(a-1)* 1000 The fractionation factor for a specific reaction is constant (Macko and Ostrom 1994), however, the magnitude of isotopic fractionation between a source and a product is a function of the extent of reaction and substrate availability. By calculating the isotopic ratios at different times in a uni-directional reaction in a closed system, the (a) of the reaction can be determined. 4 THE MICROBIAL NITROGEN CYCLE The microbial nitrogen cycle entails four primary processes: nitrogen fixation, mineralization, nitrification and denitrification. Nitrogen/fixation is the transformation of elemental dinitrogen into ammonia. In a terrestrial system there are two primary sources of fixed nitrogen. The first source is provided by the process of dinitrogen reduction into ammonia by a few select species of bacteria. The second primary source of fixed nitrogen to a terrestrial system is the application of nitrogen fertilizers. The majority of fixed nitrogen in the fertilizers is provided by the Haber process. The Haber Process reduces Nag) to ammonia gas at a specific temperature and pressure. N2(9)"’ 3H2 => 2NH3 NH3(9) Under aerobic conditions, ammonia may follow one or more of three available pathways: plant and microbial uptake, volatilization and microbial nitrification. Nitrification is the oxidation of ammonia to nitrite and nitrate (Figure 1, step 2). 5 2NH3 => N02 => N03 Nitrate produced in this step may follow one or more of the three following pathways: it may be assimilated by plant growth, leached to ground water, or under anaerobic conditions, be reduced to Nz(g) by microbial denitrification (Figure 1, step 3). The production of Nag) by denitrificaiton completes the nitrogen cycle. Fertilizer Atmospheric N2 nitrogen \Aerobic soil zone Step 1 Plant and microbial Mineralization of Volatilization of assimilation fertilizer & atmospheric of NH4 of NH4 nitrogen Nitrogen source -> NH4 TAT Step 2 Plant and microbial Nitrification of fertilizer Leaching of N03 assimilation or mineralized NH4 to ground water of N03 NH4 -> N03 I__./' Anaerobic soil zone Step 3 enitrification 3 ->N02 ->NO ->NZO - 2 y Figure 1. Microbial nitrogen cycle SOURCES OF INORGANIC NITROGEN TO GROUNDWATER The main sources of nitrate contamination of ground water have been found to be, nitrate derived from soil organic matter, human and animal waste nitrate and fertilizer nitrate (Heaton 1986). Over 90% of the nitrogen found in the surface layer of most soils is found is the form of organic nitrogen (Stevenson 1982). In many soils, the large reservoir of organic nitrogen can act as a source of nitrate through the mineralization process. Mineralization of organic nitrogen has been shown to increase with the initiation of cultivation. Mineralization of organic nitrogen is often responsible for the high nitrate concentration found in groundwater (Heaton, 1986). Elevated nitrate concentrations derived from geologic parent material are not generally considered a major source of nitrate contamination. Portions of the Fort Union geologic formation in North Dakota, Montana and Wyoming, however, are an example in which geologic sources are important. The elevated nitrate levels found in these shallow aquifers result from nitrification of high levels of ammonia derived from lignite coal (Fedkiw, 1981). The concentration of naturally occurring nitrate in groundwater is often less than 3mg/L (Fedkiw 1981). However, the agricultural practices of fertilizer application and the operation of large feed lots has resulted in an increase in the concentration of nitrate found in many rural wells to well over 10 mg/L (Follett, 1989). 8 The US. Geological Survey has noted that approximately 20% of 124,000 wells tested nation wide, exceeded the 10 mg NOg-N/ L groundwater standard. These exceedances were generally attributed to anthropogenic activities (Follett, 1989). The sudden growth of urban population into rural areas not serviced by municipal sewers has resulted in an increased dependence on single household septic systems. Throughout the rural US and Canada, septic systems are often located on top of unconfined aquifers, thus creating a direct conduit for nitrate leaching to groundwater. In many environments, sufficient nitrate has leached to the groundwater to raise concentrations above the drinking water standard (Kreitler, 1979). THE 615N OF NITRATE SOURCES TO GROUNDWATER Stable isotopic analysis may provide information leading to the determination of the sources, fate and transformation of nitrate in groundwater. Three main sources of nitrate contamination can be distinguished on the basis of 615N: soil derived nitrate (2 to 8 0/00), animal waste nitrate(10 to 20 0/00) and fertilizer nitrate (-3 to 2 0/00) (Kreitler, 1979). Over the past few decades, considerable research has been directed towards the use of the nitrogen stable isotope technique to determine the source of groundwater nitrate contamination. Soil derived nitrate has been found to be one of the main sources of nitrate found in groundwater (Kreitler, 1975). Cultivation has been found to increase organic nitrogen mineralization (Heaton, 1985) and 9 may explain why some areas have high nitrate concentration in groundwater even though fertilizers have not been used. For example, in Runnels County in Texas, groundwater collected in the vicinity of cultivated fields in which no fertilizer had been applied had average nitrate concentrations of 250 mg/L and 015N-values between 2 0/00 to 8 0/00. Dating measurements indicated that the water was less than 20 years old. Thus, 015N of the water and the lack of fertilizer application suggest that the nitrate found in the groundwater was soil derived (Kreitler, 1975). Although cultivation enhances the mineralization of organic nitrogen it is not a prerequisite for soil-derived nitrate to be found in ground water. For example, water of a confined aquifer in the western Kalahari had 015N-values between 4 and 8 0/00 . Water within this aquifer was dated at 3000 yrs SP. and predated human cultivation practices. 015N-values of the water were indicative of soil derived nitrate (Heaton, 1984). Of the three primary sources of nitrate contamination, nitrate from animal and or human waste has the most distinctive isotopic signature. Nitrate from animal waste has been shown to have 015N- values between +10 and +22 0/00 (Kreitler and Jones, 1975). Nitrate found in ground water of Grand Cayman island had 015N- values between +14 to +24 0/00. The Cayman area is largely comprised of small communities and houses with individual septic systems. This observation in conjunction with high 015N-values is indicative of nitrate contamination from animal and human waste (Kreitler and Browning, 1983). Nitrate contamination of the lronshore Formation in Texas is another example where animal 10 waste was the main source of nitrate. The 015N-values for ground water samples in this area ranged from +18 to +239 0/00 and thus, were Strongly indicative of animal waste (Krietler and Browning, 1983). The high 015N-values characteristic of animal and human waste result from ammonia volatilization, which leaves the residual ammonia enriched in 15N. Animal waste nitrate is excreted in the - form of urea which is rapidly hydrolyzed to ammonia in the environment. Hydrolysis of urea causes an increase in the pH of the waste mixture that readily allows the ammonia to volatilize. Upon volatilization, the pH is lowered and the remaining ammonia is stabilized (Krietler and Jones, 1975). Preferential release of 14N during volatilization leaves the residual ammonium enriched in 15N AS a result, nitrification of the residual ammonia results in nitrate with 15N values often greater than 10 0/00 (Kreitler, 1979). Nitrogen-based fertilizers have isotope values between ~8 to +6 0/00 (Krietler, 1979, Flipse and Bonner, 1985), although the majority of fertilizers range between -3 to +2 0/00 (Krietler, 1979). Groundwater samples taken from a housing development in Medford, New York, had 015N-values that ranged from +1.1 to 7.1 0/00 and averaged 4.3 0/00. These 015N-values are indicative of nitrate contamination from fertilizers and are probably the result of lawn fertilization. (Flipse et. al., 1984). A similar Study in the central Platt region of Nebraska found that application of commercial fertilizers was the only reasonable source of nitrate contamination. This was confirmed by 015N-values for nitrate in groundwater samples of 3.5 to 6.5 0/00 (Gormly and Spaulding, 1979). 11 ISOTOPIC FRACTIONATION IN THE MICROBIAL NITROGEN CYCLE Mineralization, nitrification and denitrification can significantly affect the isotopic composition of groundwater nitrate and thereby compromise the determination of origin based on 015N (Heaton 1984 and Delwiche and Steyn, 1970). For example, it was found that a 20% removal of the nitrate pool via denitrification can an increase the 015N of residual nitrate by 8 0/00 (Heaton, 1984). Rapid denitrification within the Chalk Aquifer in northern France left the residual nitrate enriched in 15N with 015N of 4.7 to 5.0 0/00 (Mariotti et. al., 1988), thus illustrating the influence of microbial processes on the isotopic Signature of the nitrate. The direction of fractionation may allow for the identification of the process causing isotope alteration. For example, when artesian ground water from the western Kalahari was sampled to determine the nitrogen isotopic composition, it was found that fractionation due to denitrification enriched the residual nitrate in 15N by as much as 30 0/00 (Vogel et al., 1981). Although other processes within the nitrogen microbial cycle cause isotopic fractionation only denitrification can cause such large enrichments. Mineralization and subsequent nitrification of the NH3 in soils usually produces N03 similar in 015N to the organic matter from which it was derived (Feigin et al., 1974). Due to the slow rate of mineralization there is generally only a small pool of mineralized nitrogen available for nitrification. As result the the 015N of the 12 resulting nitrate is similar to the 015N of the original substrate (Vogel et al., 1981). In environments where large reservoirs of ammonium are present, the rate of nitrification limits the extent of fractionation. The nitrate produced in this scenario is often depleted in 15N relative to the original substrate (Marriotti et al., 1988). The expression of fractionation during nitrification is often observed following the application of ammonium-based fertilizers (Kreitler, 1979). ISOTOPIC COMPOSITION OF SOIL NITROGEN There are four main reservoirs of soil nitrogen: (1) nitrate, (2) ammonium, (3) readily mineralizable organic nitrogen compounds and (4) organic compounds not usually available to plants or microbes (Feigin et. al., 1974a). The first three reservoirs provide the vast majority of the inorganic nitrogen needed for plant uptake and microbial reactions in soils. The wide range of 015N values of soil organic matter is indicative of the complexity of the soil biosphere system. A number of studies have found that there is high spatial variability in the 015N of soils (Rennie et. al., 1976, Broadbent et. al., 1980, Ledgard et. al., 1984). For example, in a small drainage basin the 015N of soil organic matter ranged from 5.0 to 7.9 0/00 (Ledgard et. al., 1984). In a Study of 015N content of two soil profiles from central Illinois, it was discovered that the 015N of the top 30cm of soil increased over the summer from 1.2 to +10 0/00(Feigin et. al., 1974). 13 The enrichment of soils in 15N is attributed to the conservation of the heavier isotope over time (Delwiche and Steyn, 1970) Since the microbial nitrogen cycle favors the consumption of 14N. Therefore, the remaining mineralizable pool of organic nitrogen and the subsequent nitrate is isotopically enriched in 15N. ISOTOPIC COMPOSITION OF PRECIPITATION On a global scale, the amount of nitrogen available to soils from precipitation is on the same of order of magnitude as the amount of fertilizer nitrogen applied to cultivated fields (Freyer, 1978). Nitrogen deposition from precipitation at the study Site is approximately 13 -15 kg/ha. Precipitation nitrogen is therefore, not a primary source of N03 in leachate. The 015N values of nitrate and ammonium in precipitation have been found to be highly variable (Macko and Ostrom, 1994). Studies that have weighted 015N values by their respective concentrations of nitrate and ammonium have shown that precipitation nitrate and ammonium are depleted in 15N relative to soil organic matter. For example, rain samples collected in Pretoria, South Africa, over a period of one year had weighted average 015N values for nitrate and ammonium of -3.5 0/00 and -3.8 0/00, respectively (Heaton, 1987). In another study, precipitation samples collected over a two-year period in Julich, Germany had mean nitrate and ammonium 015N values of -3.1 0/00 and -9.1 0/00 respectively (Freyer, 1978). 14 FIELD INVESTIGATIONS Kellogg Biological Station is located 15 miles northeast of the city of Kalamazoo in Kalamazoo County, Michigan. Two test plots at the Long-Term Ecological Research (LTER) Agricultural Ecology Site were utilized in this study: (1) lysimeter No. 2, conventionally farmed and tilled; and (2) lysimeter No. 9, conventionally farmed and not tilled. Both conventionally farmed plots were on an annual cropping system of rye-corn rotation. To gain a better understanding of the factors influencing the isot0pic transformations and fate of inorganic nitrogen in a terrestrial farming system, leachates from soil lysimeters placed in the tilled and non-tilled plots were collected and analyzed for the 015N and concentration of N03- and NH4+. Leachate from each of the two test plot lysimeters was sampled weekly from March to November 1993. Lysimeter samples were collected to determine the seasonal flux and isotopic composition of nitrate leaching from the upper soil column. The 015N of wet/dry atmospheric deposition and soil organic matter was determined to define the initial isotopic composition of these sources. SYNOPSIS The purpose of this research is to determine how processes within the microbial nitrogen cycle in agricultural soils vary seasonally and as a function of tilling practices. The Stable nitrogen isot0pic technique can provide insight into the dominant microbial 15 nitrogen cycle processes on a seasonal and spatial basis. An understanding of the influence of seasonal variation and tillage practices on the microbial nitrogen cycle and nitrogen speciation will provide insight into the the fate of inorganic nitrogen in agricultural systems. CHAPTER 2: MATERIALS AND METHODS LYSIMETER HISTORIES Table 1 provides a chronological review of cr0p rotation and chemicals applied to lysimeters 2 and 9. Lysimeters 2 and 9 were installed in May of 1990 and have been on a rye/ corn rotation since that time. They were installed with a minimum of disturbance to the soil column. Lysimeters 2 and 9 were 4 feet by 5 feet and 10 feet deep and have been conventionally tilled and not tilled, respectively. The lysimeters were accidentally fertilized at a rate of 172 lbs N./acre with ammonium nitrate (34-0-0) fertilizer in July of 1990. The lysimeters were flushed with water for several days to remove the fertilizer. There has been no fertilizer added Since. Round-up pre-plow herbicide, Dual pre-emergence herbicide, Banvel post-emergence herbicide and Difonate ZOG insecticide have been applied as needed. However, Roundup pre-plow herbicide was accidently over applied in 1991. 16 17 Table 1. Chronological Review of Lysimeters 2 & 9 Crop Rotation and Chemical Applications Year crop Insecticide Herbicide Fertilizer Notes 1990 Corn none Round-up pre plow Ammonium Lysimeters were Rye Dual pre-emergence nitrate fertilized in error Banvel post- emergence (34-0-0) 1991 Corn none Round-up pre plow none Round-up pre plow Dual pre-emergence was misapplied at ten times the usual application rate 1992 Corn Difonate Round-up pre plow none 206 Dual pre-emergence Accent 1993 Corn none Round-up pre plow none Dual pre-emergence SAMPLE COLLECTION AND PREPARATION Lysimeter samples were collected once a week from March to November 1993. Clean 500 mL Nalgene sample bottles were placed in the lysimeters for one 24-hour period each week. To minimize degradation, samples were filtered with a 40-micron filter and frozen immediately upon removal from the lysimeters. Prior to 18 distillation, the samples were thawed and the volume of sample was determined. Nitrate concentrations were determined in the (K85) lab of LC. Hedin using a Dionex ion chromatograph equipped with a membrane supressor and a conductivity detector. Soil samples from the top 5 cm of topsoil from lysimeter 2 and 9 were collected to determine the 015N of the soil nitrogen. The samples were dried in a vacuum oven and ground to a fine powder, after which they were acidified and dried again. Approximately 100 ug of dried soil was placed in a quartz tube with excess Cu and CuO. The quartz tube was then placed on a vacuum line. Upon evacuation, the tube was sealed with a torch and shaken vigorously to homogenize the metal and sample. The sealed quartz tube was then combusted at 850°C for 1 hour and allowed to cool gradually (Macko, 1981), after which the tube was cracked under vacuum to cryogenically remove all H20 and C02, The N2 created was then trapped on a molecular sieve. The 01510 of the cryogenically purified N2 was determined on a VG Prism Stable isotope ratio mass spectrometer. Wet and dry precipitation samples were collected throughout the study. Upon collection, the sample was immediately frozen and then thawed prior to distillation. Precipitation and temperature data were provided by Lolita Krievs of Kellogg Biological Station. The weekly precipitation and temperature data are averages over the 7 day periods ending on the sample collection date. 19 ISOTOPIC ANALYSIS The success of the stable nitrogen isotope technique depends on the ability to convert nitrate and ammonium to N2 without altering the 15N/ 14N ratio. For this to occur, the procedure must provide complete and separate recovery of ammonium and nitrate without isotopically fractionating the sample (Bremner and Keeney, 1966; Cline, 1975; Heaton and Collett, 1985; Velinsky et, al., 1989; Ostrom, 1992). In this procedure, ammonia is separated by steam distillation using a Labconco Rapid Kjeldahl system (Rapid Still II). The Rapid Still ll consists of a 100 or 500 mL sample flask, a water cooled condenser and a heating element. To assure complete recovery, cooling water within the condenser was kept at 5.0°C using a Neslab recirculating chiller. The distillation of ammonium was completed by first raising the pH of the sample to greater than 10 with the addition of 1mL 5N NaOH to the sample. In an earlier analysis of this method, it was found that the NaOH would cause the breakdown of organic nitrogen to ammonia, thus contaminating the sample (Bremner and Keeney, 1966). The use of MgO instead of NaOH and short distillation times was found to eliminate contamination from organic nitrogen compounds (Bremner and Keeney, 1966). Velinsky, however, found that there was little difference in the 015N of the samples when using NaOH or MgO (Velinsky et, al., 1989). It was reasoned that dissolved organic nitrogen (DON) was present in concentrations too small to affect the isotope ratio or that significant decomposition of the organic nitrogen did not occur (Velinsky et, al., 1989). In a 20 similar test, the 015N of fertilizer and rainwater where determined and were in close agreement when using either M90 or NaOH (Heaton and Collett, 1985). Therefore, based on these results, the breakdown of organic matter by NaOH should not cause contamination of samples. After raising the pH of the sample with the addition of NaOH, boiling was initiated and adjusted to achieve a collection rate of 9 mL min“. Faster or slower distillation rates led to incomplete recovery. Condensate was collected through tygon tubing, passed through an attached glass pipet which was immersed in 25 mL of 0.084N HCL acid. Quantitative recovery of ammonium from 100 and 500 mL samples was obtained within 6 and 15 minutes, respectively (Figures 2 & 3) After distillation, the condensate flask was set aside for binding of the ammonium. A second distillation for nitrate was commenced. The reduction of nitrate to ammonium was accomplished with the addition of 0.3 g of Devarda's alloy (50% Cu, 45% Al, 5% Zn) by the following reaction: 3N03' + 8Al + SOH' + 2H20 - 8Al02" + 3NH3 The Devarda's Alloy was quickly added to the ammonia-free sample, which was then distilled at a rate of 9 mL min'1 into an 25 mL 0.084N HCL acid trap. Quantitative recovery of nitrate from 100 and 500 mL samples was obtained within 6 and 15 minutes, respectively (Figure 3). To assure that complete recovery was achieved, Standards were run with a known concentration of ammonium. Once 21 the time required for quantitative recovery was established, sample distillations were conducted. 110 E. 105- g 100- 95- 8 90- g 85- ‘ . - 80- , 'I NH4 in 100 ml of DIW g 75_- o NH4 in 500 ml of DIW 70- 2 3 4 6 8 10 15 20 Time (min) Figure 2. Average Recovery During Distillation vs. Time: 20 umols of NH4+ in 100ml & 500ml of Deionized Water 22 120- b110- 0 100- S 90- u _ I. 33- o , a: . 50. '0 N03 In 100 ml of DIW g 50- 0 N03 in 500 ml of cm 40- 2 4 5 6 81015161825 Time (min) Figure 3. Average Recovery During Distillation vs. Time: 20 umols of N03" in 100ml 8: 500ml of Deionized Water Incomplete recovery of ammonium or nitrate during distillation may result in a loss of isotopic integrity (Cline, 1975; Heaton and Collett, 1985; Ostrom, 1992; Velinsky et, al., 1989). Incomplete recovery during both ammonium and nitrate distillation results in appreciable fractionation in which the distillate is isotopically depleted. Other studies have shown that upon 90% recovery during distillation of ammonium, the distillate was depleted in 15N by ~ 0.5 0/00. The fractionation was even greater during nitrate distillation, in which 90% recovery resulted in the distillate being depicted by ~ 2.5 0/00 (Heaton and Collett, 1985). In a similar Study, an isotopic fractionation factor(a) of 1.0058 i 0.0004 was determined for incomplete recovery of ammonia during 23 nitrate distillation (Cline, 1975) and agrees well with the (a) calculated in this study. As in the other Studies, there was substantial fractionation with incomplete recovery of ammonium (Ostrom, 1992; Velinsky et, al., 1989). With only 40% recovery, the distillate was depleted in 15N by ~ 3.5 0/00. A fractionation factor (a) was derived for this reaction by determining the 015N of samples of known nitrate concentration. The distillation time of these samples varied in the length causing a varying degree of fractionation. Using the information derived from this Study and a modified Rayleigh equation (Macko et al. 1986). A fractionation factor of 1.0063 was determined (Figure 4). 24 105— 95- 85- 75- 65- 55- 96 Recovery 45- 35 2 5 8 10 15 20 Time (min) Figure 4. Changes in the 015N of the Distilled NH4+ over the Course of the Distillation (Note: The 015M of the standard used was -0.910/00) After the distillation of the ammonium or nitrate was complete, 0.19 of dried zeolite molecular sieve and a teflon coated stir bar were added to the distillate. The distillate was covered and stirred for 1/2 hour while maintaining a pH of 4.5 to 5.0. Ph was maintained by adding 1 normal HCL and NazHCO3 to the distillate. Quantitative recovery was best accomplished within this pH range 25 (Figure 5). At the end of the 1/2 hour, the sieve was vacuum filtered onto a precombusted Whatman GF/C glass fiber filter. After 1/2 hour of zeolite binding, 96% of the ammonia was bound (Figure 6). To achieve quantitative recovery of the ammonia, the procedure was repeated using the filtrate from the first binding. The filters were dried under vacuum at 40°C and prepared as one sample for combustion. 120 E1003 80- 60- 40- 20- O 96 Recov Figure 5. Recovery of NH4+ Using Zeolite with Varying pH: 20 umols NH4+ in 300ml Deionized Water and 60 mg Zeolite In preparation for isotopic analysis, the dried zeolite was removed from the filter and placed in an precombusted quartz tube. In addition to the zeolite, 2 grams of Cu and 2 grams of precombusted CuO (500°C for 1 hour) were added to the tube. The quartz tube was then placed on a vacuum line. Upon evacuation, the tube was sealed 26 with a torch and shaken vigorously to homogenize the metal and sample. The sealed quartz tube was then combusted at 850°C for 1 hour and allowed to cool gradually. Prior to analysis the tube was cracked in a vacuum line to cryogenically remove H20 and C02, The liberated N2 was then trapped in molecular sieve and the 015N was determined on a VG Prism stable isotope ratio mass spectrometer. The precision of this technique is 1 0/00 or better (Table 2). 110 105- 100- 95- 90- 85- 80 I IV I. IIVVI :VT-II: 10 20 30 40 60 Time (min) 96 Bonded Figure 6. Percent NH4+ Bounded Using Zeolite with Varying Time: 20 umols NH4+ in 300ml Deionized Water and 60 mg Zeolite 27 Table 2. 015N Values for IAEA N3 and KNO3 Standards Determined Using the Sealed -Tube Combustion Technique M 1.) 4.11 0,00 2.) 4.11 °/00 3,) 4,25 0,00 Avg. 4160/00 502 0.08 0,00 Baker O 1.) 5.35 /00 Z.) 5.45 /00 3.) 5.43 /00 Avg. 5.43 0,00 502 0.07 0,00 OO CORRECTION FOR BACKGROUND The distillation technique described in the section " Isotopic Analysis " has been shown to provide quantitative rec0very of nitrate and ammonium from standards in the range of 3 to 40 umols. Nitrate and ammonium yields averaged 107.9 +/- 3.1% and 102.1 +/- 1.3%, respectively (Table 3). These values are similar to those obtained by Velinsky et al., 1989a and Ostrom 1992. Like other studies, (Velinsky et al., 1989a and Ostrom 1992), a substantial background of ammonium was discovered during the steam distillation technique. Background ammonium and nitrate derived from the Steam distillation technique averaged 0.07 umoles (Table 4) and 1.41 umols/L, respectively. (Table 5) 28 Table 3. Percent Recovery of 20 umol of IAEA KNO3 and NH4 Standards in 100 ml Deionized Water Standard Initial Abundance Final Abundance 96 Recovery IAEA NH4 20 umoles 20.12 vmdes 101 IAEA NH4 20 umoles 20.16 umols 101 IAEA NH4 20 umoles 20.33 umoles 102 IAEA NH4 20 vaIes 20.80 umoIes 104 IAEA NH4 20 umoles 20.40 umols 102 IAEA NH4 20 umoles 20.14 umoles 101 IAEA NH4 20 umoles 20.61 vmdes 103 IAEA NH4 20 umoles 20.21 vmdes 101 IAEA NH4 20 vmoIes 20.16 vaIS 101 IAEA NH4 20 umoles 20.20 vmoIes 101 IAEA NH4 20 umoles 20.34 vmoIes 102 IAEA NH4 20 umoles 20.74 vmoIes 104 IAEA NH4 20 umoles 20.95 vmoles 105 IAEA NH4 20 vaIS 20.45 vmoIeS 102 IAEA N03 20 umoles 21.56 vmdfi 108 IAEA N03 20 vmoles 22.30 umoles 1 12 IAEA N03 20 umoles 23.45 vaIeS 1 17 IAEA N03 20 umoles 21.03 vaIes 105 IAEA N03 20 umoles 21.61 vmoIes 108 IAEA N03 20 umoles 21.60 vmoles 103 IAEAN03 20 umoles 21.31 vmoIes 107 IAEA N03 20 umoles 21.81 vaIes 109 IAEA N03 20 umoles 21.25 umols 106 IAEA N03 20 umoles 21.08 vmoIes 105 IAEA N03 20 vmoles 21.38 vmoles 107 IAEA N03 20 vmoles 21.32 umols 106 IAEA N03 20 vmoles 21.64 umoles 108 IAEA N03 20 umoles 21.96 vmdes 1 10 IAEA N03 20 umoles 21.44 umols 107 IAEA N03 20 vmoIes 21.68 umoIes 108 Average 105% +/- 3.8 29 Table 4. Abundance of Background NH4 Associated with the Distillation Technique Blank # umols recovered Blk #1 0.1 Blk #2 0.1 Blk #3 0.0 Blk #4 0.1 Blk #5 0.1 Blk #6 0.1 Blk #7 0.1 Blk #8 0.1 Blk #9 0.1 Blk #10 0.1 Blk #1 1 0.1 Average 0.1 Table 5. Abundance of Background Nitrate with the Distillation Technique Blank # umols recovered Blk #1 1.8 Blk #2 2.2 Blk #3 0.9 Blk #4 1.2 Blk #5 0.9 Average 1.4 +/- 0.56 Ammonium background during the distillation processes was most likely derived from air, base (NaOH) or Devarda's alloy. In order to reduce background NH4 from the NaOH, the base was distilled and diluted back to its original volume (Ostrom, 1992). 30 Additionally, the steam distillation apparatus was cleaned after every sample run by distilling deionized water. In order to obtain the actual isot0pic Signature of a sample, the background nitrogen must be accounted for in both ammonium and nitrate distillations (Cline, 1975; Ostrom, 1992). However, in this study background contamination during the distillation of ammonium was found to negligible. The average ammonium background concentration was found to be 0.07 umoles/L (Table 4) and was too low for 015N to be determined. The release of ammonia from Devarda's Alloy during the reduction of nitrate was a concern. In blanks in which 159 of alloy was used, 7 to 10 umoles of NH3 was produced (Cline, 1975). In another study, 0.97 umoles of NH3 was produced when 0.3 g of the alloy was used (Ostrom, 1992). In this study the average background concentration was 1.41 umols of NH3 produced upon using 0.3 g of the alloy (Table 5). The isotopic composition of the background was previously found to be depleted in 15N relative to atmospheric nitrogen (Cline, 1975; Ostrom, 1992). In this study, however, the nitrate background was found to be isotopically enriched in 15N relative to atmospheric nitrogen. The 015N of the nitrate background ranged from 7.0 to 15.60/00 and averaged 12. 3 0/00 (Table 6). 3 1 Table 6. 015N of Nitrate Blanks Blank # 015N Blk # 1 1 . Blk # 2 Blk #3 Blk #4 Blk #5 Blk #6 wwwwflwo comm-raccoon dddd Average +/- 3.7 In order to obtain the isotopic composition of the sample, background must be accounted for. In this Study, background correction was accomplished by using the following mass balance equation (Ostrom, 1992): 0151‘lsam = 015Nmeas (Asam + Aback) - (515Nback * Aback) (Eq. 1) Asam where: 015Nsam = the 015N of the sample. 615Nmeas = the 015N measured. 615Nback = the 015N of the background. Asam = the abundance of nitrate or ammonium placed in the distillation apparatus (umoles of N). Aback = the abundance of ammonium associated with background nitrate or ammonium 32 The isotopic composition of the sample was obtained by subtracting the background portion of 15N from the uncorrected sample using equation 1. The background concentration and its isotopic composition were determined by analyzing deionized water with an equal amount of Devarda's Alloy as was used in the distillation of the sample (Tables 5 8: 6). Prior to the use of equation 1, KN03 standards of known isotopic composition and concentration were distilled to determine the accuracy of the procedure. After subtracting out the background by mass balance, the 015N of nitrate standards matched the projected 615N of the standard (Table 7). Table 7. 015N Correction for Background U.S.G.S. KNO3 Standard (Standard ISBN 2 180 0/00) Uncorrected Corrected 1.) 168.8 0/00 - 179.6 0/00 2.) 169.6 0/00 - 180.4 0/00 3.) 170.6 0/00 —- 181.4 0/00 Avg. 180.5 0/00 + 0.90/00 CHAPTER 3: RESULTS AND DISCUSSION RESULTS Groundwater samples from two mid-Michigan lysimeters were collected on a weekly basis from March of 1993 to November of 1993. Lysimeters 9 was untilled and lysimeter 2 was tilled. Nitrate concentrations were determined as well as the nitrogen isotopic abundances. Statistical analysis of the analytical results were completed to determine Significant relationship between variables. Finally, the analytical and statistical results were assessed to determine if possible, the feasibility of using the stable isotope technique in studying the dynamics of nitrogen cycling in a terrestrial system. Lysimeter flow rates were monitored during the same period as the sample collection. Flow rates in both the tilled and untilled lysimeters were similiar with both reaching maximum and minimum flows in the spring and late summer, respectively (Figure 7 and Appendix A). Nitrate concentrations in the tilled lysimeter were approximately twice that of the untilled lysimeter and varied in a similar manner (Figure 8 and Appendix B). The variation in nitrate fluxes was largely driven by variation in the lysimeters flow rates. (Figure 9 and Appendix C). 33 34 mmx mvNC — — MS .NC 0— MC hNC 00 MC MNC mo max m C mo I mm\@o\ mo I mmx omC mo I mmx mNC mo I MC 0 C no I NC max we I max NOC we I NC wNC #0 I mmx m. C no I max N C No I NC DOC NO I max mNC mo I max pNC we I NC e C mo I mmxbox mo I max 5.130 I NC .vNC no I NC h C no I NC 0 C mo I NC MOC no I mmx wNC v0 I NC o C EU I mmx N C In. I m9. m9. we I NC —MC MD I NC mNC mo ‘ g ' I Lysimeter 2 “ . O Lysimeter 9 10— 5_ H 0 _ _ _ _ 5 5 o 5 3 2 2 .I 45— 40— _ A _ S 0 5 O 6 6 S 5 _ w A8095=w .‘Ofi n_O 055—0}. Sample Collection Date Seasonal Variations In the Flow Rates of Lysimeters 2 and 9 figure 7 3S . I BBQ : , I 3. EB— . I SECS , .. I 8meme . I we; :8 I 8328 I 3:58 I .8328 I 83:8 I 8328 I 8328 I RENE. I mm; :3 I BE :3 I 839.5 I 8398 I m2 553 I no}. :3 I 23.88 I mm: .28 I 8:.“me I mg. {no I mas—Bo I Lysimeter 2 9 Lysimeter 9 l mmx m C #0 l mmC N C *0 l MOCmvao l mmx pmxmo l mmCmNCmo _ 0 6 .l 180* _ 0 0 2 3.28.3 «oz 440 - 420 - 400 - 380 - 360 - 340 - 320 - 300 - 280 - 260 - 240 '- 220 - 1 140 120 100- 80- 60- 40- Sample Collection Date Lysimeters 2 and 9 Dissolved Nitrate Concentrations Figure 8 36 I Lysimeter 2 P e Lysimeter 9 I nmCaNC .— I nwx NCO— . I p.288 I mmCMNCwo I maxo C mo . I mmxmox we I noCNono I max oNx #0 I max m C No I m9. N C No I nmC max #0 I nmC ONC 00 .I max —NCwo I max .v C o0 I max hoC we I max Sxoo I max vNC no I nmx h C no I nmC o C no I n9. max no I mmC ONC to I MC mNC mo 25 _ _ _ o 5 0 5 2 1| 1| M‘CNWaopbh 9826.653 .5: £52 Sample Collection Date Seasonal Variation in the Nitrate Flux of Lysimeters 2 and 9 Figure 9 37 Variations in nitrogen isotopic abundances were similar between the untilled and tilled lysimeters (Figure 10 and Appendix D). An increasing trend in the 615N of nitrate from both lysimeters occurred from spring to late summer of 1993. The 015N values ranged between -3.9 0100 in the spring to 9.4 0700 in fall (Appendix D). A decrease in 015N was observed after September 13, 1993 to the conclusion of the study on November 29, 1993 (Figure 10 and Appendix D). 815K to l MMM Iiiiiiiiiiiiiiiiiiiif 3' I Lysimeter 2 , 0 Lysimeter 9 -4... -5 NEONMMMMMMMMMMMMMMMMNMMMMMMMMMM mmmmmmmmmmmwmmosmosmosoxmmmmmmmasmm \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ l0-|DNO\\DIOOF-vv-P.¢v-OJIDNONNDNOHDIOONDIOIOF—O‘ NMDF—NO—v-NOO—NNO—wNOD—NMO—NNNN N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ mmvv-vvmmnmwwowwhhhhmwmmmmmmmo— 000000DOOOODDOOOODOOOOOOOOOOv—P Sample Collection Date Figure 10 Seasonal Variations in the 615N of Dissolved Nitrate 38 615N OF PRECIPITATION AND SOIL ORGANIC NITROGEN Precipitation and soil organic matter from the study site were analyzed for mm. The 015N of the combined wet and dry precipitation nitrate samples (3 total) had an average NW of -2.3 0/00 with a standard deviation of 1.5 0/00 (Table 8). Soil samples were collected from the top 2 cm of topsoil and from a homogenized mixture of the upper 2 feet of topsoil. Samples collected from the top 2 cm of top soil from lysimeters 2 and 9 had 015N values of 6.6 and 8.3 0/00, respectively (Table 9). These values are similar to the expected range of 2 to 8 0/00 for soil derived nitrate. Samples collected from the upper 2 feet of soil near lysimeters 2 and 9 had average 015N value of 13.6 with a average standard deviation of 1.95 (Table 9). Table 8 015N Data for Nitrate in Precipitation Date 515N 0/00 03/31 /93 -4.3 08/1 5/93 -0. 6 09/1 5/93 -2. 1 Average -2.3 39 Table 9. 815N Data for Soil Organic Matter. Lysimeter Sample 1 Sample 2 Average 515N 515N 515N 0/90 QLQQ 0/00 Ly 2 top 2 cm 6.6 Ly 9 top 2 cm 8.3 Ly210p211 15.9 11.5 137* Ly9t092fl 10.9 16.1 13.5° * Note: The 615N deterrninaitons were performed on a homogenius mixture of the upper 2 feet of the soil column. The samples were taken from two randomly selected sampling points near each lysimeter. Direct sampling of the lysimeter was considered too destructive. Samples from the top 2 cm were taken from the surface soil of each lysimeter. LINEAR REGRESSION ANALYSIS OF FACTORS CONTROLLING THE 015NOF GROUNDWATER NITRATE In an attempt to better understand factors controlling variation in the NW of the disSoIved nitrate, linear regression analysis and F-tests were performed. The 015N of dissolved nitrate ‘ from lysimeters 2 and 9 (dependent,x variable) were compared to flow rates, nitrate flux, temperature, soil moisture B horizon, soil moisture C horizon, precipitation and nitrate concentration (independent, Y variable). The variables of nitrate flux, precipitation, nitrate concentration, soil moisture B and C horizons and flow rates were not correlated with the (MN of dissolved nitrate from lysimeters 2 and 9 (R2 values for these independent variables ranged from 0.018 to 0.199, Table 10). The Null hypothesis was accepted for the relationships between lysimeter 2 and these 40 variables and lysimeter 9 and these variables with F- test values in the range of 0.01 to 4.46, o=0.05 (Table 10). Temperature had a Significant correlation with 015N values of dissolved nitrate from lysimeters 2 (R2 = 0.50, n=_ 19) and 9 (R2 = 0.45, n= 19) (Table 10). The Null hypothesis was rejected for the relationship between lysimeter 2 and temperature and lysimeter 9 and temperature with F- test values of 19.55 and 15.65 respectively, o=0.05 (Table 10). Table 10 Linear Regression Analysis (I32) and F-test (F) Data Variable Lysimeter 2 Lysimeter 9 Soil Moisture B-hor (R2) 0.0209 0.0008 Soil Moisture B-hor (F) 0.34 Q = 0.95 0.01 Q = 0.95 Soil Moisture B-hor (82) 0.0934 0.0811 Soil Moisture B-hor (F) 1.86 g = 0.95 4.46 g = 0.95 Flow Rates(F12) 0.0811 0.0175 Flow Rates (F) 2.03 p =0.9_5 0.42 g = 0.95 Nitrate Flux (R2) 0.0836 0.0093 Nitrate Flux (F) 2.10 p = 0.95 0.24 Q = 0.95 Temperature (F12) 0.5037 0.4516 Temperature (F) 19.55 Q = 0.95 15.65 g = 0.95 Precipitation (F12) 0.0641 0.0486 Precipitation (F) 1.30 p = 0.95 0.96 p = 0.95 Nitrate Concentration (F12) 0.04524 0.09873 Nitrate Concentration (F) 1.09 9 = 0.95 2.63 p = 0.95 41 Slopes of the weighted- averaged lines for lysimeters 2 and 9 were nearly identical, with a slope value of 0.0369 and 0.0396 respectively (Appendix E and Figure 11). Similar slopes in both lysimeters indicate that the processes altering the mm of the nitrate are the same in both lysimeters and are independent of the tillage practices. Delta ISN ........... .............................. 50 150 Julian Day 250 300 Figure 11 Monthly running weighted average 01615N oi lysimeters 2 and 9 DISCUSSION The oISN values of leachate collected in the early spring from both lysimeters 2 and 9 were depleted in 15N relative to other times of the year (a low of -3.9 0/00 was observed in March 31, 1993). These values may have resulted from (1) isotopic fractionation 43 during nitrification (Marriotti et. al., 1988), (2) mineralization of readily mineralizable organic matter (Feigin et. al., 1974, Tiessen et. al., 1984 and Ledgard et al., 1984) or (3)15N-depleted nitrate in precipitation (Moore, 1977; Freyer et. al.; 1978 and T.H.E. Heaton, 1987). When a large reservoir of ammonium is present, fractionation during nitrification is fully expressed and the nitrate produced is depleted in 15N relative to the ammonium (Marriotti et. al., 1988). In contrast, when a low concentration of ammonium is present, nitrification of the ammonium pool is complete and as a result, fraction during nitrification is not expressed (Feigin et. al., 1974b). The concentrations of ammonium in the leachate collected from lysimeters 2 and 9 were near or below detection limits throughout the study. As a result, isotopic fractionation during nitrification is unlikely to be the cause of the low 015N values. The readily mineralizable portion of soil organic matter has been shown to be depleted in 15N relative to the less mineralizable fraction of soil (Ledgard et. al., 1984 and Tiessen et. al., 1984). As a result, the initial mineralization of the readily mineralizable organic matter will result in the release of 15N depleted nitrate and the accumulation of 1SN enriched residual organic matter (Ledgard et. al., 1984). The depleted 15N values observed in the early spring may be the result of the initial mineralization of a readily mineralizable fraction of soil organic matter. Precipitation samples collected from KBS averaged -2.3 0/00 +/- 1.5 0/00 in 015N (Table 8). The average monthly concentration of inorganic nitrogen at the Kellogg Biological Station in 1993 was 44 53.8 M (G.P. Robertson, unpublished data on Web site http://KBS.MSU.EDU/lter/data/000_toc.htmlx). This concentration is less than 1/3 of that found in leachate from the non-tilled lysimeter and less than 1/6 of that found in leachate from the tilled lysimeter (Figure 8). This suggests that precipation nitrogen is not a primary source of N03. Due to the fact that neither of the lysimeters had recent applications of nitrogen fertilizers, and that precipitation nitrogen is not a primary source of N03, it appears likely that soil nitrogen is the primary of source of N03, The leachate-samples collected in the early spring to late summer from both lysimeters 2 and 9 show an increasing trend in (MN (3 maximum of 9.4 0/00 was observed on September 13, 1993). This trend may have resulted from isotopic fractionation during denitrification (Marriotti et. al., 1988), or mineralization of an increasingly refractory pool of organic matter that is enriched in 15N (Feigin et. al., 1974, Tiessen et. al., 1984 and Ledgard et al., 1984). As a result of warmer soil temperatures as spring progresses, microbial activity increases. Nitrate produced can either be assimilated by plants and/or the microbial population or undergo denitrification. The process of denitrification favors the lighter isotope of nitrogen and leaves the residual nitrate enriched in 15N. Enrichment of the residual nitrate will continue until the nitrate pool has been expanded or soil temperatures drop and thus decrease the rate of denitrification. The continued enrichment of the nitrate pool by denitrification, therefore, may be responsible for the increase in 015N values of leachate collected in the early spring to late summer from both lysimeters 2 and 9. 45 Another possible explanation for the observed increase in the oISN values from early spring to late summer may be the mineralization of an increasingly refractory and 15N enriched pool of organic matter. Four separate nitrogen pools have been defined in soils. They consist of (1)nitrate, (2) readily available ammonium and other nitrogenous materials, (3) less readily available organic nitrogen and (4)unavailable or unmineralizable nitrogen (Feigin et. al., 1974). During incubation studies, Feigin et. al., (1974) found that pool 1 leached out almost immediately and pool 2 produced an initial nitrate spike. As a result, the less readily available organic nitrogen, pool 3, provided substrate for the longer term production of nitrate. The readily mineralizable organic fraction has been shown to be depleted in 15N relative to the other pools of soil nitrogen (Delewiche and Steyn, 1970). As a result, the mineralization of the readily available organic matter fraction of the soil may have led to the production of 15N depleted nitrate in the early spring. The readily mineralizable organic fraction of soils is a finite reservoir. As mineralization of the readily mineralizable organic fraction of soils reaches completion, microbial mineralization proceeds to mineralize the less mineralizable and 15N-enriched organic nitrogen. Subsequent mineralization and nitrification of this soil fraction and the resulting fractionation will result in the release of 15N— enriched nitrate. Incubation studies found that the process of mineralization and subsequent enrichment of the less readily available organic fraction of soils continues until the NW of the less readily available organic fraction of soils is approximately 46 equal to the 015N of total soil nitrogen (Feigin et. al., 1974). Therefore, the increasing trend of NW values of leachate observed from early spring to late summer may be the result of the fractionation associated with mineralization and nitrification of the less readily available organic fraction of soils. The increasing trend of MN values of leachate observed from early spring to late summer may therefore be the result of either isotopic fraction during denitrification or the mineralization and subsequent nitrification of less readily mineralizable organic matter. However, it is unlikely that either is solely responsible for the observed trend. Rather, it is more likely that the observed trend is result of a combination of these two processes. The 015N values of leachate decreased from late summer to the conclusion of the study on 11/29 from both lysimeters Z and 9. This trend appears to reflect a change in the predominant microbial process controlling the NW values observed in leachate. With the onset of fall, both air and soil temperatures decreased. As a result, much of the microbial activity probably decreased. Denitrification rates have been shown to decrease significantly with cooler temperatures and are not appreciable below 4-6 degrees celsius. (Ryden, 1986; Jordan, 1989; Ruz-Jerez et al., 1994). The rates of mineralization and nitrification also likely decreased. However, site specific data suggests that these processes were still active despite low soil temperatures (G.P. Robertson, unpublished data on Web site http:/(KBSMSU.EDU/Iter/data/000_toc.htmlx). In addition, 15N- depleted plant material (harvest residuales) may have also provided 47 an additional source of mineralizable organic nitrogen. As a result, mineralization and nitrification of the 15N-depleted soil fraction, may explain the decreasing trend of (MN values of leachate observed from late summer to the end of the study. Based on this discussion, it is evident that no single source or microbial process is responsible for trends observed in the (MN values of leachate from lysimeters 2 and 9. In fact, these trends could be the result of a combination of nitrogen source and fractionation due to microbial processes. The findings of this study, suggest that soil organic matter was the primary source of nitrate to lysimeter leachate. Whereas, precipitation and fertilizer nitrogen did not appear to be appreciable sources of nitrate to lysimeter leachate. In addition, it appears likely that fractionation due to mineralization and nitrification was at least partially responsible for the trends observed in the 015N values of leachate from lysimeters 2 and 9 during the spring and early summer. Whereas, denitrification and/0r mineralization of a less mineralizable soil fraction were in part responsible for the trends observed in the summer and fall. As result, of the dynamic nature of the terrestrial system and the multitude of variables influencing the (MN values of leachate, the feasibility of using the stable isotope technique to assess origins in such an environment can be limited. It is evident from this study that influence of microbial processes on the (MN values of leachate is appreciable and it is this line of research that may be most informative. Therefore, any use of the stable isotope ' technique to determine the source of nitrogen without considering 48 the effect of fractionation may lead to questionable results. However, it is in studies such as this one where there is at least a limited understanding of the sources and processes affecting the final product (in this case the (MN values of leachate) that the feasibility of using the stable isotope technique in investigating the dynamics of nitrogen cycling in a terrestrial system is evident. APPENDIX APPENDIX A FLOW RATE DATA FOR LYSIMETERS 2 AND 9 Date Lysimeter 2 Lysi meter 9 Volume of flow Volume of flow ( Ilweek) ( Ilweek) 03/25/93 5.4236 2.086 03I31I93 3.9634 04/05/93 13.7676 2.2946 04/12/93 13.9762 5.8408 04l19/93 14. 602 1 1 .473 04/26/93 58.408 58.408 05/03/93 25.6578 26.9094 05/10/93 16.4794 16.0622 05/17/93 12.0988 1 1 .473 05/24/93 7.0924 6.6752 06/01/93 6.8838 6.0494 06/07/93 5.8408 5.8408 06/14l93 5.0064 4.3806 06/21/93 13.7676 5.0064 06/28/93 32.5416 07/05/93 24.6148 28.161 07l12/93 1 6.8966 21 .6944 07/19/93 13.3504 16.4794 07/26/93 12.516 10.8472 08/02/93 9. 1 784 8. 5526 08/09/93 6.8838 6.4666 08/16/93 6.4666 5.6322 08l23/93 5.4236 4.5892 08/30/93 4.3806 3.9634 09/06/93 3.5462 2.9204 09/13/93 1 .4602 1 .4602 09/23/93 1 .4602 12.3074 09/27/93 1 .6688 7.9268 10/21/93 40.677 40.677 1 1/29/93 5.4236 6.0494 49 APPENDIX B NITRATE CONCENTRATION FOUND IN LEACHATE COLLECTED FROM LYSIMETERS 2 AND 9 Date Lysimeter 2 Lysimeter 9 Nitrate Concentration Nitrate Concentration (MIL) (umM 0325/93 282. 9 1 72.1 03/31/93 416.8 143.7 04/05/93 398. 6 1 50.9 04l12/93 393.1 142.3 04/19/93 363.6 148.1 04/26/93 337.8 206.4 05/03/93 370.0 226.8 0511 0/93 360. 7 226.3 05l17/93 373.9 219.3 05/24/93 352.6 214.7 06/01/93 363.3 204.4 06/07I93 402.3 1 98.0 06/14/93 409.5 199.2 06/21/93 394.5 228.7 06/28/93 421 .1 259.2 07/05/93 398.4 244.1 07/1 2/93 346.3 226.9 07/19/93 387.5 256.5 07/26/93 380.7 235.0 08/02/93 366.8 08/09/93 369.2 255.3 08/16/93 370.8 213.8 08l23/93 369.5 235.8 08/30/93 333.0 2204‘ 09/06/93 323.7 191.9 09/13/93 346.4 160.5 09/23/93 342.1 238.6 09/27/93 347.3 244.0 10/21/93 345.0 245.0 11129193 34:59 245.0 i SO APPENDIX C NITRATE FLUX DATA FOR LYSIMETERS 2 AND 9 Date Lysimeter 2 Lysimeter 9 Nitrate Flux Nitrate Flux (umols/week) (umols/week) 03/25I93 1 534. 3 359 03/31/93 569.5 04l05/93 5487.8 346.3 04/12I93 5494 831. 04/19/93 5309.3 1699.2 04/26/93 19730.2 12055.4 05/03/93 9493.4 6103.1 05/10/93 5944.1 3634.9 05l17/93 4523. 7 2516 05/24l93 2500.8 1433.2 06/01/93 2500.9 1236.5 06/07/93 2349.8 1 156.5 06/14l93 2050.1 872.6 06/21/93 5431.3 1 145 06/28/93 13703.3 07/05/93 9806.5 6874.1 07/12/93 5851 .3 4922.5 07/19/93 5173.3 4227 07126/93 4764.8 2549.1 08/02/93 3366.6 08/09/93 2541.5 1650.9 08/16/93 2397. 8 1 204.2 08/23l93 2004 1082.1 08I30/93 1458.7 873.5 09/06/93 1 147. 9 560.4 09/13/93 505.8 234.4 09/23/93 499.5 2936.5 09/27I93 579.6 1934.1 1 0/21/93 14033.6 9965.9 11/29/93 1871.1 14§_2._1 APPENDIX D 015N OF DISSOLVED NITRATE Date Lysimeter 2 Lysimeter 9 Corrected 015N Corrected 015N (Cl/m) (0/00) 03/25/93 0.2 -0.3 03l31/93 -3.9 -1.5 04l05l93 -2.7 -2.3 04l12/93 -3 3.2 04I19/93 0.5 04/26/93 -0.1 1 .7 05/03/93 0. 1 -0.5 05/10/93 0.8 0.6 05/17/93 0.2 0.6 05/24/93 1.2 0.6 06/01/93 -0.5 1.4 06/07/93 1.6 06/14I93 06l21/93 4.6 4.1 06/28/93 1.8 5 07/05/93 2.9 3.2 07/12/93 4.2 3.2 07/19/93 4.8 07l26/93 6.7 6.8 08/02/93 7 5.9 08/09/93 4.3 3.7 08/16/93 5.2 7 08/23/93 3.7 5.2 08/30/93 5.5 09/06/93 3.7 5.8 09/13/93 9.4 8.8 09/23/93 8.2 09/27/93 4.7 4 10/21/93 2.3 3.2 1129/93 0 2 1.4 Note: All data corrected for background 52 APPENDIX E MONTHLY RUNNING WEIGHTED AVERAGE OF 015N Julian Ly 2 Running Monthly Ly 9 Running Monthly Date Weighted Avg. 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