AIR P O L L U T I O N D A M A G E T O C E L L M E M B R A N E S IN L I C H E N S - RESULTS OF SIMPLE BIOLOGICAL T E S T A P P L I E D IN R 1 J E K A , YUGOSLAVIA A. A L E B I C - J U R E T I C Institute of Public Health, B. Kidrica 52a, 51000 Rijeka, Yugoslavia and M. A R K O - P I J E V A C Natural History Museum, ~ Nazora 3, 51000 Rijeka, Yugoslavia (Received November 30, 1988; revised July 25, 1989) Abstract. A simple biological test developed by Pearson (1985) was applied to specimens of lichens collected within Rijeka city area to determine their plasma m e m b r a n e integrity. Two strains of lichens were tested: Parmelia tiliacea (Hoffm.) Ach,, a strain relatively tolerant, and Pannaria testacea Jorg., a strain more sensitive to air pollution. For comparison, the same test was applied to specimens of lichens collected at a coastal site in the vicinity of Rijeka as well as specimens from southern Norway. The obtained results on m e m b r a n e damage are in good agreement with SO2 concentration data (where a~ai]able) and determined lichens zones within city area. 1. Introduction Air pollution can affect plant growth resulting in abnormal or reduced N fixation, altered amino acid synthesis and provoked injuries to cell membranes (Pearson and Rogers, 1982). Lichens are very sensitive to air pollutants because their metabolism depends primarily on water and nutrient deposition from the atmosphere. The absorption and accumulation of various air pollutants, like metals (Bargagli et al., 1985; Schwartzman et al., 1987) or chlorinated hydrocarbons (Villeneuve et al., 1988) are favored by the absence of a surface cuticle. Depending on air pollution levels, city centres can be lichen deserts, surrounded by struggle zones where some kinds of lichens can still survive (though with some morphological alterations). The first study on lichens flora within Rijeka city area was published in 1902 (Schuler, 1902); however, the first classification of lichens zones is very recent (Arko, 1987). Similar studies have been conducted in Yugoslavia before (Barbalic, 1953). In this work, a simple biological test sensitive to early stages of membrane damage (Pearson, 1985) was applied to specimens of Parmelia tiliacea (Hoffm.) Ach. and Pannaria testacea Jorg. collected in the city of Rijeka. For comparison, the same method was applied to lichens collected in a coastal area approximately 25 km away from Rijeka and to those collected in southern Norway. The advantage of using biological indicators, like lichens, for monitoring air quality is that they can also indicate experienced level of pollution, i.e. long term effects (Pearson, 1985). Water, Air, and Soil Pollution 47: 25-33, 1989. © 1989 Kluwer Academic Publ&hers. Printed in the Netherlands. 26 A, A L E B I C - J U R E T I C AND M, ARKO-PIJEVAC 7 O • . . , • " • .=~ • . "~I~ • <~ 7 LLJ "i O F~ r~ • ° O D ~J0 • ° • • ° ° ° ~| ° E E I.Ll • I ~~ - Z . c.m • ° o~ z~ \° ~,= "-4r °[]DD I'-- . • . • • ° ° ° • . • • .=~ Ib • . # ~_~o== ° . ° • • i ° ° ¢) .p P o J o c,,~ F: N Y LC 27 AIR P O L L U T I O N D A M A G E TO LICHENS 2. Material and Methods Specimens of lichens Parmelia tiliacea and Pannaria testacea were collected from December 1985 to April 1986 within the city of Rijeka. Lichens from the coastal site and from southern Norway were collected in summer 1986 and 1987, respectively. Specimens of lichens from sites 5 and 10 were collected within city park areas. At least two specimens of lichens were collected at each sampling site and placed in individual paper bags for identification. The samples were kept at room temperature until testing. Due to the fact that extremely dry tissue can also considerably increase the conductivity of the leachate, all the lichen samples were placed in an humidity chamber for 2 hr, as recommended in the literature (Pearson, 1985). A covered battery jar filled with some deionized water at the bottom was used as a humidity chamber. To avoid direct contact with water, specimens of lichens were placed on highly porous discs of sintered glass. The water temperature was kept at approx. 35 °C. Parme[ia fiLiacea y = 5,5~,* O,70x "T r = 0,90 E / 100 O o 50 Cb o o ~ooO°O o 0 50 100 condudr.()aS mL-1g-l) Fig. 2. Correlation between specific conductivity (gS mL -~ g ~) and the amount of K ions leached (mg g-l) in Parmelia tilicea. 28 A. A L E B I C - J U R E T I C A N D M. A R K O - P I J E V A C After preconditioning in a humidity chamber and rinsing for few seconds with deionized water, a piece of lichen thallus (10 to 60 mg weight) was immersed in 20 ml of deionized distilled water (coonductivity 1.5 to 2.0 ixS) for 5 min. The increased conductivity of the solution, caused by electrolyte leakage (mainly K ions) from the tissue was measured using a conductometer (MA 5961, Iskra). The concentrations of K ions were determined by a flame photometer (Flapho 4, Carl Zeiss) from the same solutions. The lichens were desiccated for 48 hr and weighed, and conductivities per mass of lichen and volume of solution (~S mL lg-1) were calculated, as well as the amount of K per mass of lichen (mg g-l). The measurements were performed in triplicate, except in two cases when the amount of lichen was unsufficient to perform a third run. 3. Results and Discussion Rijeka city area with lichen zones is shown in Figure 1. Correlation coefficients Pannaria tesfacea y= 1,33x - 19,83 "T O r=0,93 E 100 0 o 0 o 50 0 / O i I 50 100 conciuct(pS mL-1 g-l) Fig. 3. Correlation between specific conductivity (IsS mL -~ g-~) and the amount of K ions leached (rag g-~) in Pannaria testacea. 29 AIR POLLUTION DAMAGE TO LICHENS between specific conductivity and the amount of K per mass of lichen tissue for both species (Parmelia tiliacea, r = 0.90; Pannaria testacea, r - 0.93) indicated that increased conductivity of the leachate is mainly due to K leakage from the tissue (Figures 2 and 3). The results of this simple biological test are shown in Figures 4 and 5. In the Parmelia tiliacea experiment, the largest specific conductivity and K content in the leachate was found in samples from sites 3, 4 and 5, thus indicating that these zones are exposed to higher air pollution levels, presumably with winter averages above 70 gg m -3, a concentration that certain Parmelia species can endure (Ferry et al., 1973). This is in good correlation with average winter concentrations obtained for period 1982-1987 (Institute of Public Health, 1982-1987). These values vary from 77 to 118 ~xg m -3 at site 4, and from 68 to 89 ~g m -3 at site 5. An unexpectedly high specific conductivity was noticed at site 7, and further attention should be paid to lichen specimens from this area. Pannaria testacea (more susceptible to S Q ) collected at sites 2, 6, 7 and 10 where the same test applied to Parmelia tiliacea had its lowest values (except for Rijeka Parme[ia fiIiacea A E (./3 [] conducfivify [] K confen, 100 E 8 1 50 I- 1 50 IIIIIII 1 IIIIIn IIIInl iiiiii1 i I site : 1 2 3 4 5 6 7 8 9 10 Fig. 4, Results of biological test applied to Parmelia tiliacea collected within Rijeka. 30 A. ALEBIC-JURETIC A N D M. A R K O - P I J E V A C site 7) - also showed increased specific conductivity and K leakage implying that this zone had experienced winter average SO2 concentrations slightly above 30 ~g m -3 (Ferry et al., 1973). We have not, however, data on SO2 concentrations at these sites but it is assumed that they are the cleanest within city area because of the largest distance from the city center a n d / o r industrial zones and, on the elevated terrain, the most exposed to dominant winds (mainly from NE directions). Samples of lichens were collected at a coastal site about 25 km from Rijeka gave different conductivities and K contents of leachates (Figure 6). Lichens collected were Parmelia perlata (Huds.) Ach., Parmelia sp., Cetraria sp. and Parmelia saxatilis (L.) Ach.. Parmelia perlata, the most sensitive species, suffered the greatest cell damage, while the most resistant, P. saxatilis, could be considered healthy. This results indicate that winter mean SO2 concentrations can exceed 30 ~g m -3, but are below 70 ~g m -3 (Ferry et al., 1973). These is in good agreement with the winter mean SO2 concentrations determined at the nearest sampling site with similar population density (Kraljevica, about 5 km away) that varies from 17 to 55 l~g m -3 The smallest specific conductivity and K content of leachate are found in samples Rijeka Pannaria fesfacea "T [] conductivity [] K content C~n 100 ~ E 100 ~ ~ E r.-o 50 site: 50 2 6 7 10 Fig. 5. Results of biological test applied to Pannaria testacea collected within Rijeka. AIR POLLUTION DAMAGE 31 TO LICHENS of three lichen species collected at a sparsely inhibited area in southern Norway (Figure 7). Lichens collected were Pseudeverniafmfuracea (L.) Zopf., Dermatocarpon sp. and another Dermatocarpon sp.. The lichens tested are known to grow in unpolluted areas and, according to the test applied, can be considered healthy. 4. Conclusions The results obtained with this simple biological test to determine cell membrane integrity are in good agreement with SO2 concentration data (where available) and determined lichen zones within city area. Though the smallest specific conductivity and K content in P. tiliacea test were found at sites 2, 6, 7, and 10, which are classified in normal lichen zones, the pronounced cell membrane damage in the more sensitive P. testacea suggests that the struggle zone could be easily extended. The unexpected results obtained with P. tiliacea for site 7 could be explained by the fact that this complex hilly area is on the boundary between the struggle zone and the normal zone, but in proximity of a small lichen desert (Figure 1), where P. testacea can still be found. Site: coastal area 25 km away from Rijeka [] conductivity [] K content tO A '3" c~I00 ¢.. CIJ , I00 ¢D E E E ::l. .4.- IX. g t~ "ID tO E gl_ ._m 50 ¢10 x ¢v 50 E ¢D r~ 0 Fig. 6. Results of biological test applied to lichens collected at a coastal site in the vicinity of Rijeka. 32 A. A L E B I C - J U R E T I C AND M. ARKO-PIJEVAC Site: southern Norway [] conductivity [11111 K content A ~c~I00 "3" 100 ~n E E x~ to (11 -d #._ tO 1.3 "c --1 to "E 50 (1/ cL tn " o rn ca. 50 09 O (D to E O Od tO o E o Fig. 7. Results of biological test applied to lichens collected in southern Norway. This method can be very useful in evaluating air pollution over vast areas when either starting a particular air pollution monitoring program or when such a monitoring program is hard to conduct. Acknowledgments The assistance of Mrs. N. Baric and Mrs. L. Vlasic in performing flame photometry analyses is gratefully acknowledged. References Arko, M.: 1987, Book of Abstracts, 3rd Congress of Croatian Biologists, Mali Losinj. Barbali~, L. J.: 1953, Glas. Biol. Sekc. Prirodosl. Drust, Ser. IIB 7, 99. Bargagli, R., Iosco, F. R and Leonzio, C.: 1985, Inquinamento 27, 33. Ferry, B. W., Baddeley, M. S., and Hawskworth, D. L.: 1973, Air Pollution and Lichens, The Althone Press, London, pp. 46-47, 340. Institute of Public Health Rijeka: 1982-1987, Annual Reports. Pearson, L. C. and Rodgers, G. A.: 1982, Phyton 22, 329. Pearson, L. C.: 1985, Atmos. Environ 19, 209. AIR POLLUTION D A M A G E TO L I C H E N S 33 Schuler, J.: 1902, Zur Flechtenflora yon Fiume, Separat-Abdruck aus Mittheilungen des Naturwissen Schaftlichen Clubs in Fiume', 1901, Jahrgang VI, Buchdrukerei R Battara, Fiume (Rijeka). Schwartzman, D., Kasim, M., Stieff, L., and Johnson, J. H. Jr.: 1987, Water, Air and Soil Pollut. 32, 363. Villeneuve, J. - R, Fogelqvist, E., and Cattini, C. H.: 1988, Chemosphere 17, 399. -