THE EFFECT OF SULPHITE AND FLUORIDE ON CARBON DIOXIDE UPTAKE BY MOSSES IN THE LIGHT

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1 New PhytoL (1974) 73, THE EFFECT OF SULPHITE AND FLUORIDE ON CARBON DIOXIDE UPTAKE BY MOSSES IN THE LIGHT BY F. INGLIS AND D. J. HILL Department of Plant Biology, University of Newcastle upon Tyne {Received 12 June 1974) SUMMARY Four mosses, Bryum argenteum, Grimmia pulvinata, Hypnum cupressiforme and Tortula muralis were exposed to sulphite, and their uptake of radioactive bicarbonate measured. About 50% reduction in ^*C uptake was caused by o.oi-o.i mm sulphite. The effect of ph indicated that SO2 (or 'H2SO3') was the active molecular species. Fluoride had little effect on ^*C uptake. INTRODUCTION Mosses are very sensitive to air pollution as has been shown by a number of workers (including LeBlanc, Skye and Gilbert, see Nash, 1973). However, there has been relatively little work on the effect of specific atmospheric pollutants on mosses. Bell (1973) found that the development of moss cover on soil was reduced during SO2 fumigations. Coker (1967) studied the effect of sulphur dioxide on epiphytic mosses and came to similar conclusions to those of Rao and LeBlanc (1966) who studied lichens, namely that sulphur dioxide caused breakdown of chlorophyll to phaeophytin and 'plasmolysis' of the chlorophyllous cells. Syratt and Wanstall (1969) studied the effect of gaseous SO2 on chlorophyll breakdown in three species of bryophytes. Although they used high levels of SO2 (mostly 5 ppm = 14,300 /ig/m-'), they found that 2 ppm (5720 /(g/m^) SO2 caused up to 2/3 of the chlorophyll to break down to phaeophytin in all three species and that 5 ppm caused the breakdown of 80-90% of the chlorophyll. More breakdown occurred when the bryophytes were under high air humidities than when under low. During exposure to SO2, Hypnum cupressiforme gained little sulphate; Metzeria furcata and Dicranosweisia cirrata gained about three times this amount. They suggested that the last three species avoided the effect of SO2 by oxidizing it to sulphate. They did not look at any photosynthetic processes. There does not seem to be any other information on the effects of sulphur dioxide or fluoride on specific cell processes in mosses. This paper examines the effect of sulphite on the uptake of carbon dioxide by mosses in the light. Hill (1971) showed that low concentrations of sulphite stopped carbon dioxide uptake by lichens in the light and the concentration required to do this seemed to depend on the tolerance of lichens to air pollution. He also found the effect was very sensitive to ph, with sulphite being most effective below ph 5. It is of interest to know whether these conclusions can also be applied to mosses. As fluoride is another atmospheric pollutant receiving considerable attention, the effect of fluoride on carbon dioxide uptake was also studied briefly. 1207

2 I2O8 F. INGLIS AND D. J. HILL MATERIALS AND METHODS Material of the mosses Grimmia pulvinata, Hypnum cupressiforme and Tortula muralis were collected from walls in the Tyne Valley to the west of Newcastle upon Tyne. Bryum argenteum was collected from the pavement of the University in the City of Newcastle. Apical portions, free from contaminating soil and debris, were removed and 50-mg samples weighed out. These were then put into 50-ml conical flasks containing 10 ml of medium buffered to ph 4.0 with 0.05 M phthalate in the case of media with sulphite, and to ph 6.0 with 0.04 M MES in the case of those with fluoride. Other ph values were obtained using buffers as in Hill (1971). The flasks were then incubated in a water bath at 15 C with fluorescent strip-lighting giving 10,000 lux illumination. After the prescribed incubation in sulphite or fluoride, 5 /zc NaH^^COs (20-50 mci/mm) in 10 A of water were added directly into the same medium and photosynthetic incorporation of the ^*C was allowed to proceed for a further 30 min. The moss was then killed and extracted in a fume cupboard by adding 5 ml of glacial acetic acid to the medium. The rapid reduction of ph drove off excess H^*C03~ and killed the cells by destroying the membranes allowing the radioactive metabolites to be released from the tissues. Without removing the moss, lo-x aliquots of the extract were pipetted onto small squares of Whatman No. i filter paper, which, after drying, were put into scintillation vials with 5 ml of toluene containing 5 g/1 PPO and 0.3 g/1 POPOP and counted on a Packard Tricarb Liquid Scintillation Spectrometer. Sulphite concenfrotion Fig. I. The cffect of sulphite concentration on carbon dioxide uptake., Theoretical lirie (aee text)., Bryum argenteum; A, Tortula muralis; D, Hypnum cupressiforme; O, Grimmia pulvinata. RESULTS Sulphite The uptake of carbon dioxide was sensitive to sulphite at 0.02 mm, when the uptake was reduced to of the control samples (Fig. i). At 0.50 mm carbon dioxide uptake was effectively stopped although there was some incorporation of ^*C. At 0.02 mm Bryum argenteum and Tortula muralis showed somewhat less sensitivity than the other two species. Bryum argenteum and Tortula muralis are known to be more resistant to air pollution in general than the other species.

3 Sulphite and fluoride effects on mosses 1209 r (a) I 000 a u 1000 r (c) (d) 4 6 ph 10 6 DH Fig. 2. Carbon dioxide uptake after treatment with and without sulphite (0.1 mm) at a range of ph values, (a) Grimmia pulvinata; (b) Tortula muralis; (c) Hypnum cupressiforme; (d) Bryum argenteum. 10 i.5r s s Fig. 3. The effect of sulphite at different ph values on carbon dioxide uptake. (Data from Fig. 2.), Theoretical line (see text). %, Bryum argenteum; A, Tortula muralis; O, Hypnum cupressiforme; O, Grimmia pulvinata., nh

4 I2I0 F, INGLIS AND D. J. HILL The relationship between ph and the effect of sulphite on the uptake of carbon dioxide was then studied. In control samples, all four species took up most carbon dioxide between ph 4 and ph 6 (Fig. 2). At ph 8 uptake was reduced to a minimal level. Similarly there was in most cases a marked reduction (possibly caused by a loss of ''*C02 from the medium) at ph 3. These two phs are probably extremes in the normal ecological range of the species. The presence of o.io mm sulphite resulted in the uptake of carbon dioxide being reduced at ph 4-5 in all cases except Bryum argenteum. In Fig. 3 the uptake of the sulphite treated samples is expressed as a fraction of that of the control samples. In this form, the data clearly suggest that B. argenteum is less sensitive to sulphite. It is interesting that at ph 6 sulphite apparently had a stimulatory effect in G. pulvinata. Table i. Carbon dioxide uptake after a ^recovery' period subsequent to treatment with sulphite Carbon dioxide taken up (as io' cpm) Moss species 30 min in sulphite 30 min in sulphite (0,25 mm) and 3 h in buffer alone Grimmia pulvinata Hypnum cupressiforme 109 ai3 Tortula muralis Bryum argenteum Table 2. The effect of fluoride treatment (24 h) on carbon dioxide uptake in three moss species Fluoride cone. Ratio carbon dioxide taken up (fluoride/no fluoride) () H. cupressiforme T. muralis B. argenteum o ,00 1, ,99 0, ,06 0,03 Table 3. Carbon dioxide uptake by Grimmia pulvinata after treatment with fluoride Fluoride cone. Carbon dioxide taken up (as 10' cpm) (mm) 2 h treatment 24 h treatment o a X 652 aoo It is important to establish whether the effect of sulphite on carbon dioxide uptake is permanent or temporary. In an experiment, some samples of moss were incubated for 30 min in sulphite and other samples for 30 min in 0.25 mm sulphite followed by 3 h in buffer alone before the introduction of H^'^COj". The results (Table i) show that G. pulvinata, Tortula muralis and Bryum argenteum had no capacity for recovery within 3 h of treatment with sulphite. In conclusion the data suggest that: (i) low concentrations of sulphite adversely affect carbon dioxide uptake in the four mosses studied; (ii) the effect is most pronounced below ph 5; (iii) B. argenteum appears to be less sensitive than the other species; and (iv) there is little or no recovery within 3 h after the removal of the moss from the sulphite.

5 Sulphite and fluoride effects on mosses 12 ii Fluoride The effect of fluoride on the four mosses was also studied. Material of the mosses was placed in fluoride solutions atph 6.0 for 2^h\ then H^^COj" was added fora further 30 min before extraction of the material. The results show that carbon dioxide uptake by the mosses was much less sensitive to fluoride than to sulphite by a factor of 100 (Table 2). In Grimmia pulvinata even 200 mm fluoride did not cause marked reduction in the carbon dioxide uptake after 2 hours incubation (Table 3). After 24 h, however, uptake was efl^ectively stopped. G. pulvinata and Tortula muralis appeared to be slightly less sensitive than Hypnum cupressiforme and Bryum argenteum. DISCUSSION The mosses studied appear to be ns sensitive if not more so than the lichens studied by Hill (1971). The most sensitive lichen, Usnea subfloridana was effected by 0.1 mm sulphite. The mosses in the present study were affected by 0.02 mm sulphite. However, the alga in the lichen may be shielded from the effect of the externally supplied sulphite by the fungal tissues and thus it may appear to be more resistant to sulphite. With regard to the effect of ph, mosses appear to be more sensitive to sulphite at acid values than lichens. In general, it would seem likely that lichens and mosses behave in a similar way towards sulphite. Hill (1971) found that Lecanora conizaeoides, a lichen known to be very tolerant to air pollution, had a much more resistant uptake of carbon dioxide to sulphite as compared with two other species which are known to be less tolerant of air pollution. Bryum argenteum, a moss characteristic of urban and polluted habitats, had a more resistant carbon dioxide uptake than three other species less tolerant of air pollution. There seems therefore to be a correlation between the effect of sulphite on photosynthesis and the sensitivity of the species to air pollution. Tortula muralis, which is pollution tolerant, occurs, in polluted areas, on the mortar of walls rather than acid parts of the wall. The ability of this species to grow on basic substrata probably accounts for most of its resistance to air pollution (Gilbert, 1968), sulphite (and sulphur dioxide) being less toxic at higher phs. In lichens a similar phenomenon occurs; for this reason. Hill (1971) selected lichens of similar ecological requirements with respect to acidity of the substratum. The absence of recovery 3 h after treatment with sulphite suggests that the mosses may have sustained irreversible damage. Although Coker's (1967) and Syratt and Wanstall's (1968) flnding that chlorophyll was broken down to phaeophytin may not be a primary effect of sulphur dioxide damage, irreversibility is consistent with the possibility that phaeophytin formation could be the cause of the effect of sulphite on carbon dioxide uptake in the present experiment. Ziegler (1972) gives data which suggest that sulphite is a competitive inhibitor of ribulose-diphosphate carboxylase with respect to carbon dioxide in isolated spinach chloroplasts. If such an inhibition were the cause of the effect of sulphite on mosses, the effect would have probably been reversible. However, as it did not appear to be reversible, the effect may not have been due solely to inhibition of this enzyme by this means. When sulphite or sulphur dioxide dissolves in water, three molecular species 'H2SO3', HSOj" and SO3" are formed. The proportions of each molecule depend on the ph of the solution. The 'HjSO,' form predominates at very low ph values, HSO3 ~ on the acid side of neutrality and SO3" in neutral and alkaline solutions. Rahn and Conn (1944) obtained evidence which showed that HSO3" was toxic to bacteria while 'H2SO3'was

6 I2I2 F. INGLIS AND D. J. HILL toxic to yeast. They drew graphs of sulphite concentration required to inhibit growth against ph. The points followed lines of constant HSOj" in bacteria and constant 'H2SO3' in yeast. Since the effect of ph on sulphite toxicity was so closely related to the dissociation of the sulphite molecule in Rahn and Conn's work on bacterial and yeast growth, it was thought possible in the present work that one particular form of sulphite might be involved in its effect on photosynthesis. In order to study as much of the data available as possible, the results for Grimmia, Tortula and Hypnum were combined. Those for Bryum were not included as they differed markedly. By analogy to the Dixon plot in kinetics of enzyme inhibition, the reciprocal of the ratio of ' *C fixation was plotted against the concentration of sulphite. A straight line drawn through the points has been transferred to Fig. I, and indicates a reasonable correspondence to the observed values. If the concentrations of the ionic forms at ph 4, 5 and 6 are calculated from the dissociation constants given by Rahn and Conn (1944), the reciprocal of the ratio of '''^C fixation can be plotted against the concentration of each form. Only on the plot with 'H2SO3' could a straight line be drawn through the points and it has been transferred to Fig. 3 and shows a reasonable fit to the points. It would seem, therefore, that 'H2SO3' is the form in which sulphite acts. (Bryum, being affected by sulphite at even lower ph levels than the other species, could only be affected by 'H2SO3'.) Falk and Gigu^re (1958) found that SO 2 dissolved in water could not be distinguished by their Raman Spectra, although other oxides and acids (e.g. SO3 and H2SO4) have different spectra. This strongly suggests that gaseous SO2 and SO2 dissolved in H2O are identical molecules. If this is so, it would be interesting to compare the concentrations of 'H2SO3' in solution required to inhibit the carbon dioxide uptake with the levels of SO2 in the air in polluted environments. About 50% reduction of ^'''C uptake was achieved at ph 5 with '"^^ sulphite which contains 640 //g SO2 as 'H2SO3' per cubic metre of solution. This figure is within an order of 10 of the levels of SO2 found in the air in areas where these mosses are affected by air pollution (Gilbert, 1968). It seems therefore as though treatment with sulphite in solution may indeed bear relation to the effects of gaseous SO2 in the air in urban environments. Gilbert (1968) found that mosses were killed by ppm sulphite at ph 3.2, ppm at ph 4.2 and not at all by 600 ppm at ph 6.6. He concluded that the most toxic form of sulphite was the 'undissociated acid'. This is in broad agreement with the results of this paper although he used much higher levels of sulphite. He was also observing long term survival after a 48-h treatment rather than any specific process. The results of treating the mosses with fluoride indicate that, in solution, only high levels may impair carbon dioxide uptake in the short term. However, LeBlanc, Comeau and Rao (1971) found that corticolous mosses were very sensitive to atmospheric ffuoride when they were transplanted up a fluoride gradient near an aluminium smelter. However, it should be mentioned that injury sustained by mosses after transplanting can be caused by other environmental factors than pollution (Bines, 1973). Lichens are apparently very sensitive to fluoride pollution (Gilbert, 1973) but there are no data at present which suggest a sensitivity to fluoride in short-term physiological experiments. REFERENCES BELL, J. N. B. (1973). The effect of a prolonged low concentration of sulphur dioxide on the growth of two moss species. J. BryoL, 7, 444. BINES, T. J. (1973). Aspects of the ecology of mosses Ulota crispa {Hedw.) Brid. Ph.D. thesis, University of Newcastle upon Tyne. CoKER, P. D. (1967). The effects of sulphur dioxide pollution on bark epiphytes. Trans. Brit, bryol. Soc, 5i 341-

7 Sulphite and fluoride effects on mosses 1213 FALK, M. & GiGuteE, p. A. (1958). On the nature of sulphurous acid. Can.J. Chem., 2^, GILBERT, O. L. (1968). Bryophytes as indicators of air pollution in the Tyne Valley. New PhytoL, 67, 15. GILBERT, O. L. (1973). The effect of airborne fluorides. In: Air Pollution and Lichens (Ed. B. W. Ferry, M. S. Baddeley & D. L. Hawksworth), pp Athlone Press, London. HILL, D. J. (1971). Experimental study of the effect of sulphite on lichens with reference to atmospheric pollution. New PhytoL, 70, 831. LEBLANC, F., COMEAU, G. & RAO, D. N. (1971). Fluoride injury symptoms in epiphytic lichens and mosses. Can.J. Bot., 49, NASH, T. H. (1973). The effect of air pollution on other plants, particularly vascular plants. In: Air Pollution and Lichens (Ed. by B. W. Ferry, M. S. Baddeley & D. L. Hawksworth), pp Athlone Press, London. RAHN, O. & CONN, J. E. (1944). Effect of increase in acidity on antiseptic efficiency. Ind. Engng. Chem., 36, 185. RAO, D. N. & LEBLANC, F. (1966). Effect of sulphur dioxide on the lichen alga with special reference to chlorophyll. Bryohgist, 69, 69. SYRATT, W. J. & WANSTALL, P. J. (1968). The effect of sulphur dioxide on epiphytic bryophytes. In Air Pollution, Proceedings of the First European Congress on the Influence of Air Pollution on Plants and Animals, Wageningen, 196S, pp Centre for Agricultural Publishing and Documentation, Wageningen. ZlEGLER, I. (1972). The effect of SO3 ~~ on the activity of ribulose-i,5-diphosphate carboxylase in isolated spinach chloroplasts. Planta {Berl.), 103, 155.

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