Environ. Sci. Technol. 2008, 42, 100–105
Uptake and Storage of PCBs by Plant Cuticles CLAUDIA MOECKEL, GARETH O. THOMAS, JONATHAN L. BARBER, AND KEVIN C. JONES* Centre for Chemicals Management and Department of Environmental Science, Lancaster University, Lancaster, LA1 4YQ, U.K.
Received March 31, 2007. Revised manuscript received October 6, 2007. Accepted October 19, 2007.
The uptake kinetics and storage of PCBs by isolated cuticles and cuticular waxes from Hedera helix, Prunus laurocerasus, and Ilex aquifolium were studied. Small chambers were used, allowing variation in plant uptake parameters to be studied by having the same air boundary layer in each chamber. During the 64 day study tri- and tetrachlorinated biphenyls generally reached equilibrium in waxes but not in whole cuticles. Differences between species were observed. Higher chlorinated PCB congeners did not approach equilibrium in either sample type. Although PCBs showed higher affinity for waxes than whole cuticles, the latter dominated the total uptake capacity on a surface area basis, because of the large amount of nonwax cuticular components. Mass transfer coefficients (MTCs) for PCB uptake (into both cuticles and waxes) indicated partition dependence up to log octanol/air partition coefficients (KOA) of 8.5-10, depending on species and sample type. For cuticles, higher MTCs occurred at the beginning of the experiment than later. This was not seen in reconstituted waxes, a difference which may be explained by the dispersion of intracuticular waxes within cuticles. For more lipophilic compounds, uptake appeared to be limited by diffusion processes, which may be influenced by plant physiology. Leaf surface area is, therefore, likely to control the ability of vegetation to scavenge these compounds from the air in many field situations.
Introduction Vegetation is a dynamic environmental compartment which takes up semivolatile organic chemicals (SVOCs) from the atmosphere (directly from the gas phase, or as particles containing bound chemical). It can play an important role in the environmental fate of SVOCs (1, 2), influencing concentrations in the air (3) and in wildlife (4) and contributing to their global cycling. Uptake from the air is the major pathway for the accumulation of SVOCs in plant foliage (5), although there are still uncertainties in understanding and quantifying the processes involved. In particular, an understanding of the kinetics of SVOC uptake by vegetation is required, as it appears that the leaves of some plant species may reach equilibrium (or at least steady state) with air during one growing season, whereas others do not (2, 6). Gas phase SVOCs need to move through the bulk air and the air-side boundary layer surrounding leaf surfaces, before * Corresponding author e-mail:
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partitioning to the plant material. On entering the leaf cuticle they may remain in the waxy and polymeric cuticular components or diffuse deeper into the leaf to reach intracellular storage compartments (7, 8). Which of these steps limits uptake depends on the properties of the compound, the plant and the environment. McLachlan (7) developed a framework for the interpretation of uptake observations that can help to distinguish between different controlling processes. The uptake of SVOCs by vegetation from the gasphase is often described using a two-resistance model, with an air side resistance due to the air boundary layer surrounding the leaves and a plant side resistance resulting from limited diffusion of compounds within the vegetation (9). For polychlorinated biphenyls (PCBs), field experiments provide evidence that diffusion through the air boundary layer limits the uptake rate and can prevent plants from reaching equilibrium within a growing season (10). On the air side, wind speed may be an important variable, since boundary layer thickness decreases with increasing wind speed. However, particularly for the less chlorinated PCB congeners, studies have also indicated the importance of plant side resistance (11, 12). Cuticle thickness and other properties (such as the crystallinity of the epicuticular waxes) may influence the role of the cuticle as a barrier (13). A simplified way of describing vegetation is required to use mathematical models for the fate of SVOCs in the environment, and this has often meant a one-compartment model to describe the plant (either by total plant weight or by its lipid content) (14). Generally, no distinction has been made between cuticular and intracellular lipids, even though the latter may not receive significant amounts of compounds entering the leaf mainly through the cuticle (15). Other leaf constituents, such as cutin, are generally not taken into account. However, experimental results show that twocompartment models, consisting of a fast equilibrating surface compartment and an internal reservoir compartment that needs much longer to achieve equilibrium, can explain uptake phenomena better (11, 16). It is still uncertain which constituents of the leaf (i.e., cuticular waxes alone, or along with other cuticular constituents) form the surface (rapidly reacting) compartment. The aim here was to study the uptake kinetics and storage of PCBs in isolated cuticles and cuticular waxes. Small, identically designed chambers were used, where the air boundary layer was fixed, to allow comparison of varying plant-side resistances with different isolated cuticle compartments from different species. PCBs were chosen, because they provide a range of vapor pressures and partition coefficients over several orders of magnitude within a single chemical family, to allow the influence of compound properties to be assessed.
Materials and Methods Preparation of Cuticles and Waxes. Cuticles were isolated from mature leaves of Hedera helix (common ivy, hereafter called ivy), Prunus laurocerasus (cherry laurel, hereafter called laurel) and Ilex aquifolium (common holly, hereafter called holly) using an enzyme method described in detail elsewhere (17). These species were chosen because detailed information about composition of cuticles and cuticular waxes is available from the literature. Figure 1a gives a schematic of a leaf section, showing which parts have been isolated by enzymolysis. Briefly, the method involved the following: 15 × 15 mm squares were cut from the leaves (avoiding the main veins) and immersed in an aqueous enzyme solution (2% pectinase and 2% cellulase in 0.001 M NaN3, buffered to pH 10.1021/es070764f CCC: $40.75
2008 American Chemical Society
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FIGURE 1. Schematic of (a) a leaf section, illustrating the parts isolated by enzymolysis in detail, (b) the experimental setup of the small chambers for the uptake of PCBs by cuticle and wax samples. 3 with 0.01 M citric acid and 0.01 M KOH). The leaf squares were placed in a reduced-pressure desiccator for 3 h to improve the infiltration of the enzyme solution. After an incubation period of 5-7 days, cuticles could be separated easily and any remaining tissue was removed carefully with a stream of deionized water. Only adaxial cuticles, where no (or very few) stomata can be found, were used for the following experiments. Cuticles were soaked in 0.01 M Na2B4O7 solution (pH 9) for 3 days with frequent solution changes to remove substances that were released from leaf tissues during enzymatic isolation and may have been absorbed by the cuticles (17). After washing with deionized water, cuticles were laid flat (with the epicuticular surface outermost) on to small, preweighed, aluminum foil squares and stored until further use in a jar containing a desiccant (silica gel). Cuticular waxes were extracted from isolated dry cuticle squares by soaking in dichloromethane (changed every 1.5 h) for 6 h with gentle automatic shaking. All extracts of the same batch of cuticles were pooled, reduced to a known volume and aliquots (each equivalent to one cuticle square) of this wax solution were applied with a syringe to preweighed aluminum foil squares of the same area as the cuticle squares. The amount of wax applied to each square corresponded to the average amount extracted from one cuticle. For the uptake experiment, aluminum foil pieces were only used if an even wax layer had formed after evaporation of the solvent, i.e. no spots or patches were visible, because varying thickness and surface morphology may influence sorption kinetics. The weight of each cuticle was determined directly, prior to the start of the uptake experiment. The weights of the wax layers were calculated as a proportion of the residue left from the wax solution equivalent to 10 wax squares, because
of the small amount of wax on each aluminum square. Unusually light and heavy dry cuticles were discarded to achieve a cuticle weight standard deviation below 10% for each species, for the entire study. Uptake Experiment. The experiment was conducted using small, uniformly designed chambers, so all samples experienced an air side boundary layer of the same thickness. This unmixed layer, where substances can move by diffusion only, was thicker than under real environmental conditions due to the sealed design of the chambers. The fixed air-side resistance allowed the effect of varying plant-side resistances to be studied (by using different cuticle compartments and plant species) as well as the influence of varying chemical properties. Contamination chambers were cylindrical glass jars (height: 45 mm, volume: 70 mL) with aluminum foil lined Teflon lids. In each chamber 2 mg of each of Aroclor 1248 and 1260, dissolved in hexane, were applied directly to the aluminum foil lid-lining (see Figure 1b for a schematic of the experimental setup). After the solvent had evaporated the chambers were sealed for three days to allow the glass walls to become saturated with PCBs prior to the start of the experiment. Three cuticle or wax squares were placed on the bottom of each chamber (to act as triplicate samples for one time point), together with one aluminum foil control sample. Chambers were stored at 20 ( 1.5 °C until sampling. The sampling points were 1, 2, 4, 8, 16, 32, and 64 days after the beginning of the experiment. Care was taken to minimize the time the chambers were opened and to keep them closed tightly throughout the experiment. Day 0 was represented by cuticles and waxes not exposed to PCBs in the chambers. At the end of the exposure period, samples were removed from the chambers and stored in solvent rinsed aluminum foil at -20 °C until analysis. Analysis. Prior to extraction, samples were spiked with 13 C-labeled PCB congeners (13C12 PCB 28, 52, 101, 138, 153, 180, 209) to assess their recoveries in the method. Samples (including blanks and controls) were Soxhlet extracted for 16 h with dichloromethane (DCM). Extracts were then rotary evaporated and cleaned on chromatography columns packed with acidified silica (8 g; 2:1 silica/H2SO4 by weight) underneath activated silica (8 g; Merck), eluted with hexane. Extracts were further cleaned by gel permeation chromatography (GPC) using Biobeads SX3 (BioRad laboratories) eluted with hexane/DCM (1:1). Finally, dodecane containing PCB 30 and 13C PCB 141 internal standards was added and the samples 12 were reduced to a final volume of 25 µL prior to GC injection. The samples were injected splitless and analyzed for a total of 34 PCBs (trichlorinated: PCBs 18, 22, 28 + 31; tetrachlorinated: PCBs 41 + 64, 44, 49, 52, 56 + 60, 70, 74; pentachlorinated: PCBs 87, 90 + 101, 95, 99, 105, 110, 118; hexachlorinated: PCBs 138, 141, 149, 151, 153; heptachlorinated: PCBs 170, 174, 180, 183, 187); octachlorinated: PCBs 194, 199, 203 on a Finnigan Trace GC-MS operated in electron ionization (EI+) mode, using selected ion monitoring (SIM). Separation was carried out on a CP-Sil8 capillary column (Chrompak/Varian, Palo Alto, CA, 5% phenyl, 95% dimethylpolysiloxane, length 50 m, diameter 0.25 mm) using helium as carrier gas. Quality Control. Recoveries of all 13C labeled PCBs were 71–122% with averages between 86 and 98% for individual congeners. No trend could be observed between the degree of chlorination and recovery rates. All reported values are blank corrected using the average concentrations of the aluminum foil control samples, but not corrected for the recovery rates of labeled compounds. PCB concentrations in control samples showed only small variations and were
