Measuring and Modeling of Plant Root Uptake of Organic Chemicals

The potential of xenobiotic organic compounds to be taken up by plant roots and transported to above ground tissue is an important consideration for phytoremediation projects and risk assessments alike. The transpiration stream concentration factor (TSCF), a ratio of the xylem concentration to the root-zone solution concentration, is the most commonly used parameter describing plant root uptake potential. There are two primary methods used to measure a TSCF. The first involves a mass balance, derived from intact plant experiments, while the second directly measures the TSCF of a detopped plant placed in a pressure chamber. Measuring a TSCF using intact plants can be problematic due to high cost, time requirements, and the difficulty in correcting for potential losses due to metabolism and phytovolatilization. The pressure chamber method for measuring TSCF has been successfully used to obviate these concerns. In this study adaptation of the basic method along with several novel techniques were developed for conducting plant uptake measurements using the pressure chamber. These adaptations and techniques amplify the robustness and scope, while increasing the overall validity of the method. Basic adaptations including, a root-zone solution sampling port, reducing excessive solution purging by using oxygen to pressurize the system, and the use of tritiated water as a conservative tracer. Measurements of several nutrient TSCFs and the use of mercuric chloride illustrating the effect of root membrane disruption and 25 death on TSCF are presented. Finally, the results of pre-exposing plants before use in the chamber along with uptake kinetics are given. Incorporating these measurements and techniques into the basic pressure chamber method lends legitimacy to the measurements made and leads to an improved understanding of the uptake process. Introduction Phytoremediation efforts and risk assessment determinations rely on the ability to determine the potential for xenobiotic organic compounds to be taken up by plant root systems and transported to foliar regions. The uptake potential is often measured using the TSCF (6, 11, 32, 51-53). A TSCF is the ratio of the concentration in the plant xylem or transpiration stream to the concentration in the solution the roots are using for transpiration (1). The usefulness of a TSCF is a result of the normalization of the amount of compound taken up by a plant to the total amount of water transpired during a plant’s exposure to that compound. Normalizing uptake to transpiration allows for linear scaling of total plant uptake to transpiration dependant factors such as exposure time, plant age, size, and climatic conditions (54). By knowing a compound’s TSCF, the total mass of compound in a plant shoot can be obtained by multiplying by the effective concentration of the exposure solution, the amount of water transpired during the exposure period, and then correcting for metabolism and volatilization losses (35). The TSCF is measured in one of two basic ways (see Chapter 4). In the traditional approach, a TSCF is estimated using intact plants grown either in soil or under hydroponic conditions. Because collection of enough xylem sap for analysis is difficult for most living plants, TSCFs are generally determined from calculations using the measured shoot concentrations normalized to the amount of water transpired during the 26 exposure period, i.e., concentration in xylem sap is deduced as the total mass of the compound in shoots divided by the volume of water transpired (2, 4, 6, 7). These calculations require that metabolism or volatilization within the plant be corrected for, but this is often analytically difficult and there are no uniform methods for performing these corrections. Furthermore, a TSCF derived from intact plants is often time and money intensive. The second method requires the use of a pressure chamber. The pressure chamber specifically addresses many of the difficulties encountered in using intact plants. The pressure chamber method consist of sealing the roots of a detopped plant (i.e., the above ground tissues are removed just above the lowest leaves) in a pressurized chamber containing a solution of a known concentration of the chemical of interest (11, 21, 53). The solution is drawn up through the roots as the chamber is pressurized and the xylem is collected and analyzed as it exits the cut stem. Ideally, the pressure is carefully adjusted to provide a constant flow rate corresponding to the transpiration rate of an intact plant of similar size without damaging the roots (Chapter 4). A TSCF is calculated from the ratio of the steady state xylem concentration to the root exposure concentration (21, 53). The primary advantage of the pressure chamber method comes from its simplicity. The reduction in operational variables tends to increase the pressure chamber’s consistency and repeatability (Chapter 4). Nevertheless, some adaptations and measurement techniques for incorporation into the basic process are proposed in an effort to increase the validity, robustness, and scope of the basic method. These adaptations include the addition of a root-zone solution sampling port for measuring xylem and solution concentrations in parity, use of pure pressurize the system 27 oxygen to reduce purging, and the use of tritiated water as a conservative tracer. The use of mercuric chloride is used to demonstrate the viability of the method, the effect of root health on TSCF and elucidate the importance of the root membranes to the uptake process. Finally, the results of pre-exposing plants before use in the chamber along with uptake kinetics information are given. Materials and Methods The method of plant cultivation along with the fundamental techniques of measuring a TSCF in the pressure chamber are thoroughly described in several previously published works (11, 21, 53, 55). The basic method used in this study consists of detopping plants just below the first cotyledonary node (lowest leaves) and removing all of the shoot tissue except for a small section of stem. Following the prescription of Hsu et al. (21) for sealing the plant into the chamber using plastic sheeting wrapped around dental impression material, tended to develop leaks over time in the area between the stem and stem plate. Therefore, the method of sealing the plant into the chamber was modified to use short sections (5 cm) of butyl rubber (tomato) or rigid platinum cured silicone (soybean) tubing of various diameters fitted over the cut stem creating a stem gasket. For rigid stems like soybeans, the gasket-covered stem can then be slipped through a hole in an inverted rubber stopper. The inverted stopper is then inserted up through the chamber’s stem plate, thus sealing it into the pressure chamber. Because of the woody nature and uniform size of soybean stems, the use of an inverted rubber stopper controls for the pressure of the chamber. Under low pressure, the system seals based on the initial insertion pressure applied to the stopper. As the pressure in the chamber increases, the 28 inverted stopper is forced further into the stem plate, increasing the pressure around the stem in a gentle, uniform manner proportional to the pressure applied. For larger more malleable stems like those of tomato plants, a stainless steel hose clamp above and below a tight fitting stem plate works well. After the tubing covered cut stem is sealed in the stem plate, the roots are immediately immersed in a stainless steel vessel containing oxygen saturated nutrient solution spiked with a known concentration of a target compound (Figure 3-1). The stem plate is then secured to the vessel with a threaded collar and the tip of a disposable pipette is affixed over the cut stem and under the tubing. The chamber is pressurized (~150 kPa) using compressed oxygen resulting in a xylem flow rate of approximately 70% of the plant’s previous day average transpiration rate. The pressure difference between the roots and xylem typically used in the pressure chamber falls within the reported range of measurements for intact plant root and xylem differential pressures (56). Chamber Adaptations. The pressure chamber apparatus Figure 3-1 consists of a stainless steel container with a threaded lid and stem plate. Modifications made to the pressure chamber device used in previous uptake studies (11, 21, 53) include a root-zone solution sampling port and stem plates with various diameter holes. Multiple stem plates with various size holes allow the fit of plant stems from a large age range as well as from different species without excess manipulation. The root-zone sampling port consists of a 1/4” (6.35 mm) stainless steel SwagelokTM bulkhead fitting attached to the lower end of the chamber with a teflon coated septum seated into the fitting by a 1⁄4’ (6.35 mm) cap. The addition of a sampling port facilitates paired measurements of the xylem and solution 29 concentrations in real time. The ability to sample the root solution in parity with the xylem sap is particularly important for volatile and very hydrophobic compounds whose concentration might be expected to change over the experiment due to surface sorption