The impact of air pollution on the integrity of cell membranes and
Transcription
The impact of air pollution on the integrity of cell membranes and
Arch. Environ. Contam. Toxicol. 24, 455-460 (1993) A R C H I V E S OF Environmental Contamination a n d Toxicology © 1993 Springer-Verlag New York Inc. The Impact of Air Pollution on the Integrity of Cell Membranes and Chlorophyll in the Lichen Ramalina duriaei (De Not.) Bagl. Transplanted to Industrial Sites in Israel Jacob Garty 1, Yuval Karary, and Joseph Harel Department of Botany and Institute for Nature Conservation Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Abstract. The impact of air pollution on the integrity of cell membranes and chlorophyll in the lichen Ramalina duriaei was studied. The lichen was transplanted from a relatively unpolluted site in Israel to a highly polluted area for a period of 10 months. The seasonal variation of the percentages of Mg as detected with the aid of scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX) on/in the cortical cells of the lichen was compared with changes in the chlorophyll integrity as expressed by the ratio OD 435 nrrdOD 415 nm. The rate of damage of air pollution to cell membranes in the lichen was compared with the increase of S as detected on the surface of the lichen thalli retrieved from industrial sites. The present study indicates that the electric conductivity parameter reflecting the integrity of lichen cell membranes was found to express the cellular damage caused to lichen thalli transplanted to a steel smelter and to oil refineries. Symptoms of damage to cell membranes are detectable in R. duriaei long before any indication of damage becomes apparent in the photobiont chlorophyll. Magnesium seems to represent a significant leakage from intracellular sites of the thallus. The accumulation of sulfur on/in the cortical cells of R. duriaei indicates that the biomonitoring sites at the Haifa Bay are contaminated by SO 2. During the past two decades several studies emphasized the feasibility of lichens as effective biomonitors (Ferry etal. 1973; Martin and Coughtrey 1982; Arndt et al. 1987; Nash and Wirth 1988; Galun and Ronen 1988). Lichens are very sensitive to SO2, fluorides, ozone, nitrogen oxides, peroxyacetyl nitrate (PAN) and heavy metals. Topics studied extensively in the context of air pollution and lichens include the rate of respiration (Baddelely et al. 1972), photosynthesis (Showman 1972; Richardson and Puckett 1973; Canaani et al. 1984), chlorophyll fluorescence (Kauppi 1980), chlorophyll content (Henriksson and Pearson 1981), decrease 1To whom correspondence should be addressed. of ATP (Kardish et al. 1987; Garty et al. 1988) and changes in the levels of endogenous auxin and ethylene (Epstein et al. 1986). The bleaching of lichen thalli as a result of the degradation of chlorophyll is one of the most obvious indications of damage by airborne pollutants (Nash 1971; Ronen and Galun 1984; Garty et al. 1985, 1988; Silberstein and Galun 1988). Ronen and Galun (1984) extracted the photosynthetic pigments from the lichens by the immersion of the thalli in dimethylsulfoxide (DMSO). They suggested the ratio of optical density at wavelengths of 435 nm and 415 nm as a reliable parameter for the estimation of chlorophyll degradation. The advantages of DMSO as a solvent for the extraction of photosynthetic lichen pigments are that the extraction is simple, rapid and complete, and the extract is easily stored in the cold without degradation. A simple test to check whether the plasma membrane enclosing lichen cell membranes enclosing lichen cells is normal is to place a piece of excised lichen thallus in distilled deionized water for two or three minutes (Simon 1974; Puckett et al. 1977). Pearson and Henriksson (1981) showed the effect of SO2 on membrane leakage in three lichens. In Evernia prunastri, increased leakage occurred at all SO2 levels from 0.32 ppm and above. The conductivity values obtained after the exposure of this lichen to 0.32, 100 and 300 ppm SO 2 were found to be 10.04, 12.25, and 300 Ixmho/g/ml, respectively when immersed for 2 min in deionized distilled water. Similar findings were reported by Pearson and Rodgers (1982), Alebic-Juretic and Arko-Pijevac (1989), Rope and Pearson (1990), and Silberstein (1990). These studies were laboratory experiments or investigations performed under field conditions in sites where SO2 was the major contaminant of the air. The major objectives of the current investigation were to study environmental conditions during different seasons of the year and to determine the integrity of chlorophyll as expressed by the OD 435 nm/OD 415 nm ratio under these conditions. The integrity of cell membranes as expressed by changes of the electric conductivity in doubly distilled water of a sensitive lichen in its native unpolluted habitat is used as the control. An additional objective of the study was to determine whether changes in the integrity of chlorophyll and of cellmembranes are comparable in the case of lichens transplanted J. Garty et al. 456 A HaZorea site, in September and December 1989 and February, April and June 1990 as fresh material ("in situ" in Tables 1-3). / N Extraction and Assessment of the Integrity of Chlorophyll The chlorophyll of lichens sampled from the sites in Haifa Bay and from the control site (re-suspended as well as in situ material), was extracted overnight in the dark in 3 ml of dimethylsulfoxide (DMSOMerck Product, analytical grade). The ratio of chlorophyll a to phaeophytin a (OD 435 nm/OD 415 nm) was determined as previously described (Ronen and Galun 1984) using a Varian spectrophotometer, model 460. I 5 km . 2" 1 Assessment of Damage to Cell Membranes by Measurement of Changes in the Electric Conductivity in Doubly Distilled Water 4. Batches of in situ lichen material, re-suspended thalli from the control site, as well as transplanted thalli retrieved from the Haifa Bay sites were transported to our laboratory at Tel Aviv University. They were rinsed for several seconds in a doubly distilled water, kept in a laboratory for 24 h, and divided into samples of 1 g each. Lichen samples were immersed in 100 ml doubly distilled water for 5 min. The electric conductivity of the water was measured with an electric conductivity meter (Radiometer Copenhagen, type CDM, 2e). 1, Energy Dispersive X-ray Analysis (EDX) of the Thallus Surface Fig. 1. The study area: (1) HaZorea forest (control site); (2) the agricultural village, Kefar Bialik, Haifa Bay; (3) the steel smelter, Kiryat Happlada, Haifa Bay; (4) the oil refineries, Haifa Bay to polluted areas from different periods of time. Changes in the integrity of chlorophyll and cell-membranes in lichens exposed to contaminated sites against changes in the elemental composition of the surface of the thallus were also investigated. Materials and Methods The fruticose lichen Ramalina duriaei (De Not.) Bagl., which grows on twigs of carob trees (Ceratonia siliqua L.), was collected throughout the study period from a site in the HaZorea Forest (site 1, Esdraelon Valley, northeast Israel), deemed 'clean' in terms of air pollution (Garty and Fuchs 1982; Garty et al. 1985). In September 1989, about 200 detached twigs covered by R. duriaei were transferred to three different monitoring stations in the Haifa Bay area (Figure 1), a region subject to the heaviest industrial pollution in Israel. One of the sites, Kefar Bialik (site 2), is an agricultural village surrounded by several industrial plants, including two fertilizer factories (Haifa Chemicals Ltd. and "Deshanim"). Another site is near the steel smelter Kiryat Happlada, in the vicinity of Kibbutz Kefar Massaryk, south of the city of Akko (site 3). Lichens were transplanted also to the vicinity of several oil refineries (site 4). Concurrently, some of the twigs carrying lichens were re-transferred to the original carob trees for use as resuspended control specimens. Fresh thalli were picked in December 1989, February, April and June 1990, and compared with the material transplanted to the Haifa Bay (which was retrieved simultaneously). In addition, in situ lichens were collected directly from carob twigs at the Lichen samples from Haifa Bay and the control site were rinsed carefully in doubly distilled water and then air-dried for 2 d. The thalli were mounted on sample stubs, coated with carbon using a Polaron coating unit E S100 and examined in a Jeol 840A Scanning Electron Microscope (SEM) operating at 25 kV. Analyses were performed directly in the SEM, using the Link System 860. Elements were analyzed with a ZAF-4 program. The voltage for the energy dispersive analysis was 25 kV. Results of EDX using the ZAF-4 program are given as percentages of each of the 12 analyzed elements on the cortex of thalli from different sites and are evaluated with Duncan's multiple range test. For that purpose, ~/arcsin P/100 transformation was used first in order to obtain the normal distribution. The percentages of Mg and S detected on the lichen cortex are reported in this paper. Results The OD 435 nrrdOD 415 nm ratios of the samples from the exposure sites are presented in Table l. In previous studies, values of 1 . 4 5 - - - 0 . 0 3 (Ronen and Galun 1984) and 1.42 --+ 0.03 (Garty et al. 1985) were obtained from R. duriaei in the unpolluted HaZorea forest site. In these studies the OD 435 nm/OD 415 nm ratio was found to be the lowest (0.56 --- 0.03) in acidified solutions (Ronen and Galun 1984) and 1.03 -+ 0.22 in R. duriaei transplanted to a polluted site in Tel A v i v (Garry et al. 1985). Results obtained by the analysis following the examination of the R. duriaei lichen material indicate that the OD 435 nm/OD 415 nm ratio, expressing the integrity of the chlorophyll in the photobiont partner, proved to be the greatest in April (Table 1). A significant degradation of the chlorophyll was observed in the lichen material exposed to Integrity of Cell Membranes and Chlorophyll in Lichens 457 Table 1. Chlorophyll degradation expressed as OD 435 nrrgOD 415 nm ratio in the thalli ofRamalina duriaei collected at the study area. Values are based on 10 replicates. Values in each vertical column followed by the same capital letter and values in each horizontal line followed by the same small letter do not differ significantly at p = 0.05 by Duncan's multiple range test Site number 1 Site description HaZorea Forest (control site), in situ material Sep. 1989 Dec. 1989 Feb. 1990 Apr. 1990 June 1990 2 3 4 HaZorea Forest (control site), re-suspended material The Agricultural village Kefar Bialik (Haifa Bay), transplanted material Kefar Massaryk near the steel smelter (Haifa Bay), transplanted material The Oil Refineries (Haifa Bay), transplanted material -+ 1.45 0.05 BC -+ 1.44 0.04a BC -+ 1.45 0.02a -+ 1.45 0.05 B --+1.42 0.03b B -+ 1.44 0.03a -4-1.45 0.05 BC -+ 1.41 0.03b C -+ 1.47 0.09a +--1.45 0.05 A -+ 1.42 0.03b A -+ 1.43 0.06a -+ 1.45 0.05 B -+ 1.41 0.03b C -+ 1.45 0.01a B B B A B -+ 1.52 0.02a A -+ 1.42 0.02a C + 1.50 0.03a A -+ 1.43 0.01a B -+ 1.52 0.03a A -- -+ 1.46 0.09b A -+ 1.11 0.14b B -+ 1.52 0.02a A -+ 1.39 0.03a C Table 2. Electric conductivity of doubly distilled water measured five min after thalli of Ramalina duriaei collected at the study area have been soaked in. Data are given in mS m -~ .* Values are based on 10 replicates. Values in each vertical column followed by the same capital letter and values in each horizontal line followed by the same small letter do not differ significantly at p = 0.05 by Duncan's multiple range test Site number 1 Site description HaZorea Forest (control site), in situ material Dec. 1989 Feb. 1989 Apr. 1990 June 1990 2 3 4 HaZorea Forest (control site), re-suspended material The Agricultural village Kefar Bialik (Haifa Bay), transplanted material Kefar Massaryk near the steel smelter (Haifa Bay), transplanted material The Oil Refineries (Haifa Bay), transplanted material -+0.37 0.10c -+0.31 0.12c -+0.95 0.30c -+0.96 0.12b -+2.41 0.38a B B A B B -+0.07 0.00d C -+0.31 0.09c -+0.14 0.04cd C -+0.31 0.04c -+0.29 0.05c B -+0.99 0.22ab -+0.47 0.12b C -+0.86 0.21b -+ 1.00 0.45a C -+ 1.08 0.17a B B A B C +0.75 0.12c A -+0.79 0.15c A -- -+4.55 0.49a A -+3.00 0.24b A * milliSiemens/meter the industrial sites at Haifa Bay (sites 3 and 4), which was retrieved in June 1990. The integrity of cell membranes in R. duriaei as expressed by changes in the electric conductivity of water is shown in Table 2. The findings reveal that at all biomonitoring sites the lowest electric conductivity values were obtained in the winter (February 1990) whereas a seasonal peak was recorded in June 1990. The highest electric conductivity values were observed in the lichens retrieved in June from the industrial sites (sites 3 and 4) at Haifa Bay. The elemental analysis of the thallial cortex of R. duriaei samples retrieved from Haifa Bay revealed that the percentages of magnesium increased gradually during the entire period of the experiment (sites 2, 3 and 4, Table 3), whereas the control thalli (site 1) were found to contain low Mg values on the thallus surface during the entire study period. The percentages of S detected on the thallus surface of the control material were quite similar during the period September 1989 (0.22 -+ 16%)-June 1990 (0.20 -+ 0.08%), whereas the accumulation of this element in the lichen exposed in the agricultural village increased gradually during the period September 1989-April 1990. A similar trend was observed at the two other sites at the Haifa Bay: the percentages of S on the thallus surface of the lichens retrieved from the steel smelter reached at 458 J. Garry et al. Table 3. Percentages of magnesium detected on the cortex of Ramalina duriaei collected at the study area and analysed with the aid of SEM combined with EDX. Values are based on analyses of 30 thalli. Values in each vertical column followed by the same capital letter and values in each horizontal line followed by the same small letter do not differ at p = 0.05 by Duncan's multiple range test Site number 1 HaZorea Forest (control site), Site description Sep. 1989 Dec. 1989 Feb. 1990 Apr. 1990 June 1990 material HaZorea Forest (control site), re-suspended material -+0.08 0.06 A -+0.08 0.05b A ---0.07 0.04b A -0.10 0.06b A -+0.08 0.06c A -+0.08 0.06 AB ±0.11 0.05ab A ---0.07 0.05b B -+0.10 0.05b AB ±0.07 0.04c B in situ the end of the experiment 0.41 --- 0.15%, whereas corresponding values of S as detected on the surface of the lichen thalli retrieved from the oil refineries in June 1990 was 0.37 --- 0.14%. Discussion Our study indicates that symptoms of damage to cell membranes of either the lichen mycobiont or the photobiont partner, or both, are detectable in R. duriaei transplanted to polluted sites long before any indication of damage becomes apparent in the photobiont chlorophyll. In the current experiments, the degradation of chlorophyll occurred in thalli transplanted to the steel smelter only towards the end of the investigation. On the contrary, Wetmore (1988) stated that the algae of the thallus in lichens are the first to be damaged in areas with air pollution and that the first indication of damage is discoloring and death of algae, quickly leading to the death of the entire lichen. A pollution-resistant lichen species, Parmelia sulcata, was reported by von Arb (1987) to be the only one encountered in the most polluted parts of the city of Biel, Switzerland. The chlorophyll content was found to be the highest in lichen at four central and very polluted parts of the city whereas in the periphery, three to five times lower. That and similar reports (von Arb and Brunold 1990; von Arb et al. 1990) may be compared with reports of the relatively tolerant lichen Xanthoria parientina (Silberstein and Galun 1988; Silberstein 1990), which like P. Sulcata is well adapted to polluted areas. As for R. duriaei, the decrease in the OD 435 nm/OD 415 nm ratio upon transplantation to polluted sites indicates that the lichen is rather sensitive to pollution and is therefore endandgered throughout the whole Haifa Bay region. That conclusion is supported by our earlier report of a significantly low OD 435 nm/OD 415 nm ratio in indigenous R. duriaei thalli collected on a certain peak of Mount Carmel, Israel (Garry et al. 1985). Our findings 2 3 4 The Agricultural village Kefar Bialik (Haifa Bay), transplanted material Kefar Massaryk near the steel smelter (Haifa Bay), transplanted material The Oil Refineries (Haifa Bay), transplanted material -+0.08 0.06 B ±0.11 0.07ab B -0.15 0.08a A -4-0.18 0.1 la A -- -+0.08 0.06 C -+0.10 0.07a BC -+0.15 0.08a B -0.14 0.09b B ---0.23 0.15a A -+0.08 0.06 C -+0.14 0.08a AB ±0.14 0.08a AB ±0.12 0.07b B ±0.16 0.07b A regarding the use of R. duriaei as a bioindicator of air pollution indicate that lichen thalli taken from a relatively clean site undergo drastic changes upon transferring to polluted sites, mainly because of the impact of pollution and not because of the transplantation itself. As for the changes in cell membrane permeability as reflected by the increase of electrolyte leakage, the electric conductivity parameter was found to express successfully the cellular damage caused to R. duriaei thalli transplanted to the steel smelter and to the oil refineries at Haifa Bay. Magnesium seems to represent a significant leakage from intracellular sites of the thallus, indicated by a detectable accumulation on the lichen surface. Because the control thalli exhibited a different pattern of Mg accumulation under similar climatic conditions prevailing in the whole study area, we suggest that the accumulation of that element on/in the cortical cells is connected with ion leakage from internal parts of the thallus, followed by deposition on the surface cells, as detected by the SEM + EDX. Such leakages appear to result from environmental stress caused by the contaminated air prevailing in the Haifa Bay area. Magnesium, which accumulated on/in the cortical cells of R. duriaei may have been derived from chlorophyll degradation. Boonpragob and Nash (1990a) found that during summer periods at a polluted site in Los Angeles, chlorophyll and net photosynthesis declined substantially in Ramalina menziessi and that the percentage of phaeophytins increased in proportion. Leachable Mg, Ca, P, Na and K were found to increase in this lichen during the summer (Boonpragob and Nash 1990b). The authors stated that at least a part of the elevated rate of leachable Mg in R. menziessi exposed to polluted conditions derived from the degradation of the chlorophyll in the algal cells of this lichen. Lichens in their natural environment may be subjected to repeated cycles of drying and wetting. Loss of a proportion of Integrity of Cell Membranes and Chlorophyll in Lichens their solutes each time they are wetted could have an important cumulative effect (Simon 1974). Such cycles can occur quite often in the Mediterranean climate even in winter time. Buck and Brown (1979) showed that desiccation caused a significant loss of intracellular K and Mg in some lichen species, and that the loss may be related to water available in the natural habitat. These findings may explain the gradual increase of Mg on the thallus surface especially in the lichens transplanted to Haifa Bay and retrieved in June 1990. Essential ions were reported to be leached from lichens cells upon exposure to chemical contaminants, e.g., SO2 and heavy metals, under laboratory conditions (Puckett 1976; Puckett et al. 1977; Nieboer et al. 1979; Goyal and Seaward 1982; Brown and Beckett 1983; Beckett and Brown 1984; Silberstein 1990; Garty and Delarea 1991), or in field studies (Fuchs and Garty 1983; Garty et al. 1985; Alebic-Juretic and Arko-Pijevac 1989; Silberstein 1990; Boonpragob and Nash 1990b). As for the accumulation of S on/in the cortical cells of R. duriaei, the results of our investigation indicate that the biomonitoring sites at Haifa Bay are contaminated by SO2. High concentration of SO2 in the air in the surroundings of lichens was previously proved to cause elevated concentrations of S in lichens (Gilbert 1969; Hawksworth 1973; LeBlanc and Rao 1973; Pyatt 1973; LeBlanc et al. 1974; Takala et al. 1985; Garty et al. 1988). High SO2 concentrations were reported to cause damage to agricultural plants in Haifa Bay area (Chaim et al. 1973; Naveh et al. 1979). High S concentrations, detected in R. duriaei thalli retrieved from the Haifa Bay area, reflect the contamination of the air by SO2 emitted by heavy industrial plants. That contaminant seems to be connected with the membranal damage in transplanted lichen thalli and the resultant increase of electric conductivity; high values occurred especially in the lichen material retrieved from the steel smelter and the oil refineries in,June 1990. Recently, it was reported that the margins of the Haifa Bay area have been exposed to acid rain with a pH range of 4.4 4.7 and high concentrations of SO42(Shamay et al. 1990). Because no regular instrumental monitoring of air pollutants is currently conducted in the Haifa Bay area, it is recommended that a program be established to monitor contaminants and the integrity of chlorophyll and cell membranes in lichens transplanted to industrial sites. This information would document the relative significance of aerial distribution, and the fallout, of industrial pollutants. 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