ITNI was originally utilized to assess nitrogen deposition in agriculturally influenced ecosystems . Agricultural plants and cultivated crop species such as corn, rye, and wheat among others, have traditionally been utilized as ITNI study species. Additionally, the ecosystems under investigation were heavily influenced by applied nitrogen fertilizer. The latest investigation into ITNI, as of 2014, has been in peat bogs of Germany . This investigation is considered semi-natural, the peat bogs in question have been drained due to surrounding agricultural and livestock operations and therefore represent an ecosystem that is highly influenced by agricultural processes and is altered significantly by human intervention . To date, no investigators have used the ITNI method to measure Ndeposition in arid or semi-arid regions. The application of the critical load concept in semi-arid ecosystems is hampered by uncertainties in estimating dry and gaseous nitrogen deposition . Coastal sage scrub habitat is especially prone to underestimations of N loading because of arid conditions and the difficulty involved in measuring dry deposition and gas exchange. Since rainfall is usually a minor component of nitrogen loading in coastal sage scrub habitats,containers size for raspberries new methods of measuring nitrogen deposition must be employed. Underestimation of critical loads can misinform environmental policy and regulation with potentially negative effects on ecosystem restoration efforts.
Therefore, my overall goal is to use the ITNI method to better assess and account for all nitrogen uptake pathways by coastal sage scrub and provide a more accurate measurement of atmospheric nitrogen deposition in arid and semiarid ecosystems. The main objective of this chapter was to test the concept of isotope dilution and nitrogen deposition measurement using the ITNI system. It was intended that the ITNI treatments identify the movement and level of equilibrium of 15N in the module system. A specific objective was to test the suitability of the ITNI method in a non-agricultural setting, in which we investigated Coastal Sage Scrub habitats and utilized non-agricultural species such as common California native and Mediterranean invasive plants. By employing coastal sage scrub species in the ITNI method, we can demonstrate the potential to determine species-specific nitrogen deposition rates which would be useful in plant community landscape deposition mosaics of non-agriculturally influenced systems. Our two ITNI study sites were selected based on previous nitrogen deposition data produced by traditional measurement techniques . Both ITNI sites lie along a well-described nitrogen deposition gradient in southern California . My main study site, denoted as “Riverside”, is located at the United States Department of Agriculture Forest Service Pacific Southwest Research Station situated 0.6 miles south of UC Riverside. This location is representative of an urban area receiving elevated levels of nitrogen deposition.
ITNI modules were installed in a fenced workyard containing native and invasive plant species, including California buckwheat,California sagebrush, summer mustard , red brome, and ripgut brome among others. Using conventional methods for deposition monitoring, previous studies have estimated annual N deposition at Riverside to be 14 kg N ha -1 yr -1 . Motte Rimrock Reserve, denoted as “Motte,” is located in the city of Perris, California. Perris is a thirty two square mile city of approximately 70,000 residents. Motte Reserve is managed by the University of California Natural Reserve System which offers protected habitat for California native animal and plant species. Motte contains Riversidian coastal sage scrub and riparian woodlands in addition to native and invasive grasslands. Representative Riversidian species include California buckwheat, California sagebrush, brittle bush, white sage, and black sage. Summer mustard, red brome and ripgut brome are representative Mediterranean invasive plants at the site. Motte, with its buffer of protected habitat surrounded by rural and suburban style developments, represents a moderate nitrogen deposition site . ITNI modules were placed in the middle of the reserve, in an open field adjacent to the reserve manager’s office and dormitory. This site was selected for its lack of obstructing structures or trees and its visibility by Reserve Staff to protect valuable ITNI components. Using conventional methods for deposition monitoring, previous studies have estimated annual N deposition at Motte Reserve to be 12.1 kg N ha -1 yr -1 .Seeds of invasive plants were harvested from Sycamore Canyon Wilderness Park, an open-area preserve in the city of Riverside.
Vegetation at Sycamore Canyon consists mainly of Mediterranean invasive plants such as, ripgut, red brome, summer mustard, Russian thistle, among others, with small pockets of native species such as California buckwheat and California sagebrush. Sycamore Canyon was chosen as a seed collection site because of its proximity to both the ITNI study sites. Additionally, ecotypes present this Sycamore Canyon were representative of species adapted to both experimental sites. Invasive species used in the ITNI experiments included Bromus rubensin Deployment 1 and Hirschfeldia incanain Deployments 2 and 3. Bromus rubens and Hirschfeldia incana seeds were harvested from mature plants within Sycamore Canyon during September 2013. Approximately 600 seeds from Bromus rubens were carefully removed with nitrile gloves and placed into a labeled paper bag. Hirschfeldia incana seeds were harvested by selecting senescing mature plants and removing twigs that contained seed pods. The twigs were placed into a labeled paper bag, where the seed pods were broken open by gloved hand to expose the seeds. Seeds of California buckwheat , the native plant used in the ITNI experiment, were acquired from S&S Seeds . The California Buckwheat seeds were shipped in one-ounce sealed plastic bags and, upon arrival, were transferred into a paper bag for storage. Since California buckwheat is a winter germinating species, seeds were stored in a refrigerator for two weeks to increase germination success by breaking any dormancy induced by warm weather conditions. After refrigeration, the seeds were transferred into a petri dish for sepal removal. California buckwheat seeds, or kerns,big plastic pots are actually covered by sepals, or leftover petals from the flowering body that senesce around the seed. The function of the sepal is to protect the seed until germination conditions are correct for growth. To further induce germination, the sepals were removed from the seeds using tweezers. Seeds were propped up on the petri dish, blunt side down, and pushed with the tweezers until the seed was extracted from the sepal material. Successfully extracted kerns were checked for viability by examining for breaks in the seed coat and using a light source to verify that the endosperm inside the seed was still viable. Seeds with breaks in the seed coat and those that did not have a plump endosperm were discarded. Those that passed the viability inspection were placed in another petri dish for germination. A 1 mg/ml solution of gibberellic acid was used to increase the germination rate of California buckwheat, which typically exhibits less than a 65% success rate . The seeds were placed in a petri dish with a single paper towel folded into the bottom of the dish. Approximately 100 seeds were placed on top of the towel and then the gibberellic acid solution was pipetted on top of the seeds and left to soak in the solution overnight. After twenty four hours, the seeds were rinsed with DIW to remove any remaining gibberellic acid and then transferred onto water-dampened paper towel. Approximately 50 seeds were transferred onto each petri dish, using tweezers to place seeds equidistant apart along the entire bottom petri dish. After seeds were evenly distributed, a second paper towel was folded to the shape of the petri and gently laid on top of the seeds. This towel was also damped with Nanopure water without leaving standing water in the petri dish. Once this was complete, the lids were loosely placed on the dishes to help maintain moisture and placed on an east-facing windowsill to provide sunlight. Water was added to the dishes every morning to ensure proper moisture in the dish for germination. On cloudy days a 60W soft-light desk lamp was used to provide additional lighting.
California buckwheat seeds typically took about 3 weeks to fully germinate into small seedlings while summer mustard germinated in about 3 days. Therefore, native seedlings were always started a month prior to an ITNI deployment to improve seedling survival and viability. For Deployment 1, red brome was sown directly into the modules and therefore there was no pre-ITNI germination period for these seedlings. Red brome was sown directly into modules because the time to maturity, and subsequent senescence was much shorter than the other species used in the ITNI modules. ITNI modules were constructed from General Hydroponics Water Farm Modules , obtained from Discount Hydroponics in Riverside, California. Each module utilized a two reservoir system in which a solid growth medium, in this case sand, was placed in a small reservoir with a perforated bottom. To prevent loss of sand, the bottom of the sand reservoir was lined with 80 µm Nitex fabric. The sand reservoir drained into a lower liquid reservoir that contained the liquid growth media and isotope tracer .These tubes joined in the liquid reservoir via a junction piece at the bottom of the liquid reservoir. At the top of the tube system, a watering ring delivered liquid throughout the upper sand reservoir. Water lift was generated when air was pumped into the bottom of the water lift tube . A single DC-powered DC-20 model 12 Volt air compressor, controlled by a Hydrofarm 7 Day Dual Outlet Digital Timer, provided compressed air to the ITNI modules. The pump was powered by AC power at Riverside, and at Motte we used a deep-cycle lead-acid battery that was kept charged by a 30 Watt, 12 Volt, Unlimited Solar Off-Grid Solar Panel. To discourage microbial growth, the ITNI module components were rinsed with a dilute bleach solution, scrubbed with a bottle brush and then rinsed copiously with deionized water. The liquid reservoirs were labeled with volume markings, which were used in conjunction with a clear level-tube connected to the front of the liquid reservoirs to maintain a constant liquid level in the modules. To protect this tubing, and prevent nitrogen deposition into this extra orifice, a plastic sheath was used to cover the tube when not in use for adjusting and checking water levels. All ITNI modules were assembled in a greenhouse to prevent contamination from prolonged outdoor exposure before deployment. The liquid reservoir was filled with nine liters of deionized water. The air lift, water lift tubing, and watering ring were assembled with all junctions connected. Nitex was laid in the sand reservoir and was marked where the opening for the air and water lift was present. At this mark a small X was cut into the Nitex to allow the air and water lift to perforate, but not allow extra space for sand to escape into the liquid reservoir. The sand reservoir was stacked into the liquid reservoir container. The assembled air and water lift was slid through the hole in the Nitex, about half way up the shaft of the assembly. This assembled piece was then installed, with the air and water lift junction piece passing through the opening in the sand reservoir, and the assembly resting with the Nitex on the perforated sand reservoir bottom. This action left the watering ring just below the height of the sand reservoir when assembled. Once the ITNI hardware components were in place, #16 Silver Sand was added to the sand reservoirs. Each module was filled to two inches below the top of the sand reservoir to reduce sand losses to wind and by transport. The watering ring was adjusted to sit just a millimeter or so above the sand; this was done to prevent any “splattering,” of nutrient solution from bubbles in the water lift and to prevent interference by birds drinking water from under the ring . The sand used in the ITNI modules was composed of sub-angular quartz grains with an effective size of 0.5 to 0.7 mm and is a type commonly used in sand-blasting operations. The #16 Silver Sand was baked at 150 to 200 °C before commercial packaging, resulting in a moisture content of < 0.1% by weight and an essentially sterile media.