Environ. Sci. Technol. 2007, 41, 2978-2984
Transplants of Flavoparmelia caperata (L.) Hale were used to test possible relationships between chlorophyll a fluorescence (CaF) and ambient atmospheric conditions (temperature, precipitation, SO2 and NOx levels). Portions of the same thalli collected in a pristine site (A) of the Trieste Karst were exposed at that site, as the control, and in four other sites (B-E) in NE Italy, near to pollution monitoring stations. These sites had been selected in order to provide similar two by two climatic conditions (sites B,C: more humid; D,E: drier) and air pollution load (sites B,D: low; C,E: high). Before exposure and after 43 and 90 days of exposure, CaF measurements were carried out in the laboratory under controlled conditions. A classification of meteorological and pollution parameters recorded during exposure substantially confirmed the differences between site couplets. After 90 days, samples from sites A (control) and B (with very low pollution load) showed only slightly reduced NPQ, qN, Fo, and Fm values. Samples from site D, with medium air pollution load, and sites C,E, with high air pollution loads, showed proportionally greater variation for most of the CaF parameters. A highly significant correlation was found between NPQ, qN, Fm, and NOx pollution but not with SO2 or O3. Effects of NOx on lichens and possible action mechanisms are discussed. The results strongly suggest that CaF measurements of lichen transplants can be a valid tool in biomonitoring studies.
airborne pollutants, since to date the few in situ studies give rather contradictory results (9-12). These have probably arisen as a result of the limited attention given to the influence of other factors, such as the intrinsic variability in the samples and their different photoadaptation status and capacity. The variability in a lichen population is usually rather high, depending in part on the morphological variation that is related to the thallus life cycle. In parmelioid lichens, old parts are generally photosynthetically less active than young ones (13). Further complexity is given by the development of sexual reproductive structures or vegetative symbiotic diaspores, such as soredia (13), and isidia (14), that determine a different ratio between the two components of the lichen symbiosis, myco- and photobiont, modifying the chlorophyll content (14). In transplant studies, the selected material is generally morphologically homogeneous, but usually only a few measurements are taken for each sample (9). Since CaF measurements are carried out on a small area (typically ca. 0.2 cm2), they can give very different values if they are carried out in different parts of a thallus (15, 16). The influence of the light regime of the microsite colonized by the lichen is also very important. It has been demonstrated recently that thalli collected on different sides of the same tree trunk significantly differ in their light-harvesting capacity, repair systems, and chlorophyll content (17, 18), in part due to photoadaptation processes. The performance of these thalli, when transplanted to different habitats, may considerably vary, with low light adapted thalli suffering photoinhibition more frequently and intensively than high light adapted thalli (19). The CaF parameters of these two groups of thalli would also differ greatly over time, since recovery may be slow (19, 20). Finally, the seasonality of the same photoadaptation processes (17, 21) should be considered, as during the year there is a drastic modification of both light spectrum and regime caused by tree canopy formation, persistence, or fall-down (22), and not all lichens react in the same manner to these changes (23). Neglected so far in biomonitoring studies, this process might also contribute to obscure the variation induced by pollutants. This study was aimed at identifying quantitative relationships between the variation in CaF and ambient atmospheric conditions using lichen transplants. In order to avoid the above-mentioned shortcomings, great care was taken to use homogeneous material. This was exposed in highly standardized conditions close to pollution monitoring stations.
Introduction
Experimental Section
Chlorophyll a fluorescence (CaF) is the emission of part of light energy absorbed by molecules of the photosynthetic apparatus, particularly the Photosystem II (PSII). Derived CaF parameters may provide diagnostic information on the state of the photosystems (1), being related to the action of environmental factors (e.g., light stress, and water availability) (2, 3), and specific phytotoxic molecules (e.g., O3, SO2, several herbicides) (4, 5). CaF has often been used for biomonitoring purposes using vascular plants as target organisms; this approach is based on studying the effects of specific environmental pollutants on auto- and/or allochthonous (transplanted) material (6, 7). More recently, this technique has been extended to lichens, among the most widely used biomonitors of air pollution (8). By comparison with vascular plants, however, our knowledge of the factors influencing lichen CaF is not fully satisfactory, particularly in the case of
The Species. Flavoparmelia caperata (L.) Hale is a widespread, epiphytic lichen quite common throughout the mildtemperate regions of North America and Europe. Its biology is relatively well-known (13, 24), and it is often used in biomonitoring studies (e.g., refs 25 and 26), being moderately resistant to air pollution (27). Collection and Pretreatment of Samples. Wet thalli, about 15 cm in diameter, were collected from northerly exposed bark of ash trees (Fraxinus ornus L.) in a pristine wood (Percedol, Trieste, Italy; A of Figure 1), clean from air pollution (28). In the laboratory the material was left to dry out at room temperature overnight, and then it was carefully cleaned from debris, photographed, and numbered. Five groups of four samples each were prepared: one, to be used as control, consisted of four randomly selected samples taken from different thalli, whereas the remaining four groups consisted of the equitably distributed portions of four entire thalli. On each sample, two measuring points were selected, one on a marginal lobe, at 0.5 cm from the margin, and the
Effects of Ambient NOx on Chlorophyll a Fluorescence in Transplanted Flavoparmelia caperata (Lichen) MAURO TRETIACH,* MASSIMO PICCOTTO, AND LAURENCE BARUFFO Dipartimento di Biologia, Universita` degli Studi di Trieste, Via L. Giorgieri 10, I-34127 Trieste, Italy
* Corresponding author phone: ++39 040 558 3886; fax: ++39 040 575079; e-mail:
[email protected]. 2978
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 8, 2007
10.1021/es061575k CCC: $37.00
2007 American Chemical Society Published on Web 03/08/2007
FIGURE 1. Study area. Site lettering as in Table 1. other near to the center, at 3.5 cm from the margin; both were indicated in the photograph. Exposure Sites. Five sites (A-E) distributed in an area of ca. 80 × 60 km in NE Italy (Figure 1) were selected for exposure. Site A is the collection site, and material exposed there was used as the control. Sites B-E are small wooded gardens located in four urbanized areas with different traffic densities, each of them hosting a monitoring station that provided climatic and pollution data. The site choice was based on a comparison of the 40 pollution monitoring stations active in the study area. Sites C and E are among those sites with the highest air pollution load; they are located in two cities, Udine and Trieste, with rather different climatic conditions (29), the former lying ca. 50 km inland and the latter facing the Adriatic Sea. Sites B and D are among the sites with the lowest air pollution load and are climatically close to sites C and E, respectively; site B is located in S. Giovanni al Natisone (ca. 15 km from Udine) and site D in Lignano Sabbiadoro (on the Adriatic Sea). In this way, the couplets B,D and C,E were expected to offer similar air pollution load and sources, and couplets B,C and D,E similar climatic conditions during the transplanatation experiment. Further information on the sites is given in Table 1. Sample Exposure. Lichen samples were exposed for 90 days from May 17, 2005 at 3 m above ground on the northerly exposed side of deciduous trees (Table 1), being tied there with 0.05 mm nylon threads of 7 × 7 cm plastic nets (mesh ) 1 cm). During exposure, air temperature, humidity, precipitation, solar irradiation, wind speed and frequency, and concentrations of SO2, NO2, NO, and NOx (also O3 in sites C,E) were recorded, with the exception of the control site. CaF Measurements. These were carried out before and after 43 and 90 days of exposure with a pulse-amplitudemodulation fluorimeter Mini-PAM (Walz, Effeltrich, Germany), applying standard protocols (see, e.g., ref 1; abbreviations as in Table 2; photochemical (qP) and nonphotochemical (qN) quenching of variable CaF calculated as in ref 30). The samples were left to rehydrate for ca. 4 h in a closed chamber filled with wet paper, immersed for 2 min in distilled water, and replaced in the chamber at 20 °C, 10 µmol photons m-2 s-1 for 40 h. They were dark adapted for 10 min, immersed in distilled water, gently hand shaken, and positioned with
the upper surface at 90° under the measuring fiber optic. The modulated red light was turned on to obtain the minimal CaF level (Fo). A 0.8 s saturating light pulse (ca. 8000 µmol photons m-2 s-1) was applied to obtain the maximum level of CaF (Fm), and the maximum photochemical quantum yield of PSII (Fv/Fm) was calculated. Soon after, the white actinic light (175 µmol photons m-2 s-1) was turned on to record the so-called Kautsky effect. Once the peak was achieved, saturating light pulses were applied at 60 s intervals during actinic illumination to determine the transient maximum emission yield (Fm′). After 5 min, the emission of CaF at the steady state was measured, and then the actinic light was turned off. At the end of the measurements, each sample was left to dry out at room temperature in dim light (
