A proposed rapid test for susceptibility to gaseous fluorides

A PROPOSED

RAPID TEST FOR SUSCEPTIBILITY GASEOUS FLUORIDES

TO

A. W. DAVlSON, A. MARSLAND & W. E. BETTS

Department of Plant Biology, The University, Newcastle upon Tyne, Great Britain

ABSTRACT

A method, based on the use of cut leaves and fluoride solutions, is described for the rapid screening of plants .for susceptibility to gaseous fluoride and for investigating aspects of the physiology and pathology of fluoride damage. The initial experiments are used to develop a standard technique while, in later experiments, variations due to leaf age, position and environment are examined. Most of the species used were of known susceptibility. The results of the test are compared with the effects of gaseous fluoride and the practical problems are discussed.

I NTRODUCTION

Species and varieties of higher plants differ enormously in their reaction to gaseous fluorides and many efforts have been made to rank species in order of susceptibility. The field has recently been reviewed by the National Academy of Sciences (1971). Usually ranking is carried out by field observation near sources or by fumigation with known concentrations of gases but both these methods have inherent drawbacks, such as lack of control of interfering factors (field observations) and cost (fumigation). During preliminary work on varietal differences in cereals and pasture grasses it became clear that it would be useful to have a rapid screening test for selecting varieties before embarking on a fumigation programme. This paper is an account of experiments on a screening test using excised leaves and fluoride solutions. The basis of the test is as follows. When a plant is exposed to HF the gas enters the leaves and moves in solution in the transpiration stream to the margins or tips where it concentrates and produces a characteristic pattern of necrosis (Treshow & Pack, 1970). A similar phenomenon occurs when a cut leaf is partly immersed in a dilute fluoride solution ; the element enters the transpiration stream, 269 Enriron. Pollut. (7) (1974)--© Applied Science Publishers Ltd, England, 1974 Printed in Great Britain

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A . W . DAVISON, A. MARSLAND, W. E. BETTS

is concentrated and causes damage. This suggests that susceptibility could be measured in terms of the reaction of cut leaves to fluoride solutions.

MATERIALS AND METHODS

The source of plant material is specified in appropriate parts of the text. All plants raised in the laboratory were grown at a temperature of 22 + 2°C, in a daylength of 18 h and 8500 lux (Phillips H L R G Horticultural Lamps). All testing was carried out under the same conditions. Tissue to be analysed was dried at 105°C, ground in a Casella or Backman mill and analysed using a Corning specific ion electrode (Cooke, 1972). Analar grade chemicals were used throughout.

RESULTS

Establishment of a standard procedure Mature, non-senescent leaves of an Iris germanica cultivar, Ligustrum vulgare and Poa annua growing on the campus of the University of Newcastle were used to establish a standard procedure. Batches of leaves were placed in 300 ml Mono polystyrene cups containing a range of fluoride solutions (0, 5, 10, 25, 50, 100 pgF/ml as Analar NaF) with about 10 m m of the base (Iris and Poa) or petiole (Ligustrum) immersed in the solutions. The cups were placed in a growth cabinet, then after 24 h the leaves were rinsed and transferred to similar cups containing deionised water. Tissue damage was recorded up to 48 h after removal from the fluoride solutions, then the leaves were washed, sectioned, dried and analysed. There were clear differences in the reactions of the three species. Ligustrum showed no damage or change in appearance in any solution but Poa and Iris developed a dull, water-soaked appearance at the tips in concentrations of fluoride greater than 5 and 10 pg/ml respectively. The symptom was most easily seen by transmitted light. Eventually the leaf tips of Poa and Iris desiccated and necrosis spread towards the base. Forty-eight hours after the start of the experiment both Poa and Iris developed a general chlorosis in all the solutions (including controls) which was probably due to senescence. Iris germanica is usually regarded (Borsdorf, 1960; Bolay & Bovay, 1965) as being very sensitive, while Ligustrum wdgare is more tolerant (Bossavy, 1965; D~issler et al., 1972) to fluoride. The percentage of leaf area which developed necrosis (Fig. 1) was positively related to the concentration of fluoride in the bathing solution. Poa developed the greatest degree of necrosis while Iris was intermediate between Poa and Ligustrum. Analyses (Table 1) showed that Iris and Ligustrum had taken up and transported the fluoride. The Poa leaves were too small to analyse satisfactorily. Iris showed a

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271

SUSCEPTIBILITY TO GASEOUS FLUORIDES

3C-

o

Fluoride ~Jg/h~l

Fig. 1. Percent leaf area damaged after partial immersion in various concentrations of fluoride. Vertical bars indicate 95 % confidence limits: • Poa annua; • Iris germanica; A Ligustrum

vulgare. TABLE 1 ESTABLISHMENT OF A STANDARD PROCEDURE. FLUORIDE CONCENTRATIONS ( . u g F / g DRY WX) IN LEAVES PARTIALLY IMMERSED IN FLUORIDE SOLUTIONS. ANALYSES ARE MEANS OF DUPLICATE DETERMINATIONS

Fluoride in solution (ltg/ml)

Part of leaf (cm)

Species Iris germanica

Ligustrum vulgare

0

tip 0-5 5-10 10-15

68 10 4

whole leaf 54

5

0-5 5-10 10-15

11l 9 5

81

10

0-5 5-10 10-15

391 12 7

116

25

0-5 5-10 10-15

644 140 32

169

0-5 5-10 10-15 0-5 5-10 10-15

2972 875 137 2175 776 514

50

100

228

364

272

A.W. DAVISON,A. MARSLAND, W. E. BETTS

marked gradient in fluoride from the tip downwards. However, it was also found that the control leaves contained substantial concentrations of fluoride, probably from domestic coal fires (Davison et al., 1973), so only leaves from rural areas or those grown in fluoride-free atmospheres were used in later experiments. In view of the tendency for some species to senesce rapidly after cutting, and because of the high within-treatment variance, the duration of later experiments was minimised and at least ten replicates were used.

Length of exposure to fluoride and development of symptoms An experiment with barley and maize was used to investigate the effects of varying the length of exposure to fluoride solutions. Plants of Zea mais (cv. White Horse Tooth) and Hordeum vulgate (cv. Zephyr) were cultivated on John Innes potting compost in a growth cabinet. Mature, nonsenescent leaves of three-weeks-old plants were exposed to fluoride solutions for either 8 or 24 h before transfer to deionised water and then recorded for 48 h. Dark green, water-soaked areas visible by transmitted light developed after about 8 h exposure, then damage spread in a patchy manner towards the base of the leaves. Damage was much more pronounced in leaves exposed for 24 h than for 8 h. Between 8 and 24 h after the start of the experiment the damaged areas became

7£

8

3e

B

50 Fluoride jug/ml

100

Fig. 2(a). Percent leaf area of Zea mais (cv. White Horse Tooth) damaged after partial immersion in various concentrations of sodium fluoride for different lengths of time. Vertical bars indicate 95 ~o confidence limits: • 8 h contact with fluoride; • 24 h contact with fluoride.

RAPID TEST FOR SUSCEPTIBILITYTO GASEOUS FLUORIDES

273

70--

-g

35

m~

50

Fluoride .ug/ml

100

Fig. 2(b). Percent leaf area of Hordeum vulgare (cv. Zephyr) damaged after partial immersion in various concentrations of sodium fluoride for different lengths of time. Vertical bars indicate 95 ~ confidence limits: • 8 h contact with fluoride; • 24 h contact with fluoride. desiccated and senescence in the form of chlorosis started in some leaves after about 40 h. The percentage of the leaf area which developed necrosis is shown in Fig. 2(a) and (b). Necrosis was more pronounced in maize than in barley and increased with the length of exposure to the fluoride solutions. In the case of barley, which is usually rated as relatively tolerant of fluoride, an exposure time longer than 8 h was necessary to cause significant damage. This suggested that where more resistant species are concerned, long exposure might be necessary to cause damage.

Sources of variation within species (1) Leafage: The effects of H F are known to depend upon leaf age (NAS, 1971) so in a third experiment batches of young (just reaching full expansion) and mature (fully expanded but not senescent) leaves of Zea mais (cv. White Horse Tooth) were collected from pot-grown plants and exposed to fluoride solutions for 24 h. The percentage necrosis was recorded 48 h after treatment, then the leaves were sectioned, dried and analysed. There was greater development of necrosis (Fig. 3) in mature than in young leaves. This could have been due to a difference in uptake and translocation or to a difference in tissue sensitivity. Analysis of the leaf tips (Fig. 4) showed that there

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A . W . DAV1SON, A. MARSLAND, W. E. BETTS

7(3

35

50 Fluoride ,ug/ml

100

Fig. 3. Percent leaf area of young and mature leaves of Zea mais (cv. White Horse Tooth) damaged after partial immersion in various concentrations of sodium fluoride. Vertical bars indicate 95 ~ confidence limits: • young leaves; • mature leaves. 6(3

E

~ 3C o~

0

10

100 10(0) Fluo-kJe m leaf t~s, ug/g

I

1ODOO

Fig. 4. Percent leaf area of young and mature leaves of Zea mais (cv. White Horse Tooth) damaged by sodium fluoride solutions plotted against the fluoride content (#gF/g) of the leaf tips (0-5 cm): • young leaves; • mature leaves.

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275

were higher concentrations in the young leaves and therefore that the difference lay in the susceptibility of the tissues. (2) Compound leaves: In a fourth experiment two species with compound leaves were tested. The species were soybean (Glycine max cv. Hawkeye) and lucerne (Medicago sativa). Mature, non-senescent leaves of four-weeks-old pot-grown plants were cut and the petioles immersed in about 10 mm of fluoride solution. The small size of the luce/'ne leaves made supporting them difficult and several fell into the solutions during the experiment. Because of possible contamination, the lucerne leaves were not analysed. Lucerne was also prone to rapid senescence. Lucerne showed no visible damage apart from a little marginal necrosis on the middle leaflets of leaves exposed to 100 pgF/ml. Soybean, on the other hand, developed typical water-soaked areas and necrosis in concentrations above 50 pgF/ml. In 100 p g F / m l d a m a g e first appeared on the margins of the middle leaflets, then spread inwards. When about 30 ~o of the middle leaflets was necrotic, damage appeared on the margins of the lateral leaflets. After 48 h about 70/°~ of the middle leaflets and 18 ~o/ of the lateral leaflets was necrotic. The different behaviour of the leaflets was also apparent in the greater concentration (Fig. 5) of fluoride in the middle leaflets.

0

,50 Fluoride ~ / m l

100

Fig. 5. The fluoride content (#gF/g) of middle and lateral leaflets of Medicago satit'a after partial immersion in various concentrations of sodium fluoride: • middle leaflets; • lateral leaflets.

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A . W . DAVISON, A. MARSLAND, W. E. BETTS

(3) Effects of leaf environment: It is well established (Zimmerman & Hitchcock, 1956; Treshow & Pack, 1970) that the effects of gaseous fluoride vary with environmental conditions, particularly temperature and humidity, so an experiment was conducted in which the leaf environment during exposure was altered in two simple ways. The tips (25 ram) of a batch of mature barley (cv. Zephyr) leaves were smeared with a thin layer of petroleum jelly immediately before testing while other leaves were placed inside a thin-walled polythene bag during exposure to the fluoride solutions. Both treatments had pronounced effects on the development of damage, the percentage necrosis and fluoride uptake (Fig. 6, Table 2).

7C

0

~ide

50

ucj/ml

100

Fig. 6. Percent leaf area of Hordeum vulgare (cv. Zephyr) damaged after partial immersion in various concentrations of fluoride under different conditions. Vertical bars indicate 95 ~ confidence limits : • control leaves ; • leaves enclosed in a polythene bag during test; • leaf tips (0-2'5 cm) smeared with petroleum jelly.

The tips of the jelly-covered leaves remained green, healthy and undamaged but the area immediately adjacent developed the typical water-soaked appearance and eventually became necrotic. This was associated with a change in distribution of fluoride in the leaf in that the zone of maximum accumulation (Table 2) was shifted from the tip to the necrotic areas. Overall, covering the tips with jelly slightly reduced the percentage of necrosis and also the maximum fluoride concentration.

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R A P I D TEST F O R S U S C E P T I B I L I T Y T O G A S E O U S F L U O R I D E S

TABLE 2 THE EFFECTS OF LEAF ENVIRONMENT ON FLUORIDE UPTAKE. FLUORIDE CONCENTRATIONS (pgF/g DRY WT) IN LEAVES OF nordeum vulgate CV. ZEPHYR AFTER PARTIAL IMMERSION IN FLUORIDE SOLUTIONS. ANALYSES ARE MEANS OF DUPLICATE DETERMINATIONS

Fluoride in solution (ltg/ml) 0 5 10 25 50 100

Part of leaf (cm) tip O-5 5-10 10-15 0-5 5-10 10-15 0-5 5-10 10-15 0-5 5-10 10-15 0-5 5-10 10-15 0-5 5-10 10-15

Control 10 <5 <5 460 --867 241 114 1497 510 114 2768 1647 634 3212 122l 350

Enclosed in Leaf tip covered polythene during with petroleum test jelly 6 <5 <5 246 11 <5 411 28 <5 1214 111 5 1591 262 21 3650 867 211

15 0 0 333 232 20 183 284 -645 816 53 794 1773 226 1459 1887 909

Enclosing the leaves in a polythene bag d u r i n g e x p o s u r e to the fluoride solutions caused a drastic reduction in the per cent necrosis c o m p a r e d with the c o n t r o l s b u t had a m u c h smaller effect on fluoride a c c u m u l a t i o n (Fig. 6, T a b l e 2).

Comparison o f species and varieties In a final series o f experiments a n u m b e r o f species and varieties o f different leaf shape, size a n d c h a r a c t e r were tested. Cereals: Maize, barley a n d wheat are k n o w n to differ in their susceptibility to a t m o s p h e r i c fluoride so in the next experiment their tolerance to fluoride solutions was c o m p a r e d . M a t u r e leaves o f three-weeks-old plants o f Zea mais (cv. W h i t e H o r s e Tooth), Hordeum vulgare (cv. Z e p h y r ) a n d Tritieum aestivum (cv. M a r i s H u n t s m a n ) were tested. The percentage o f necrosis is shown in Fig. 7. Maize was most, a n d wheat least, susceptible to the solutions; barley was intermediate. This parallels the susceptibility o f the species to a t m o s p h e r i c fluoride ( G u d e r i a n et al., 1969; N A S , 1971). Three cultivars o f barley, Zephyr, Sultan a n d Julia, were used to examine variation within a species. Necrosis d e v e l o p e d in all three varieties; the variance was high b u t there were significant differences between t h e m (Fig. 8). Z e p h y r a p p e a r e d to be the m o s t susceptible with Sultan intermediate a n d Julia the least

278

A . W . DAVISON, A. MARSLAND, W. E. BETTS

70-

O

50 Ruoride ug/ml

tOO

Fig. 7. Comparison of the percent leaf area of Zea mais (cv. White Horse Tooth), Hordeum vulgate (cv. Zephyr) and Triticum aestivum (cv. Maris Huntsman) damaged after partial immersion in various concentrations of sodium fluoride. Vertical bars indicate 95 % confidence limits : • Zea mais ; • Hordeum vulgate; • Triticum aestivum.

60-

I

a~

0

50 FkJorK]e ug/r~

100

Fig. 8. Comparison of the percent leaf area of three varieties of Hordeum vulgare damaged after partial immersion in various concentrations of sodium fluoride. Vertical bars indicate 95% confidence limits: • cv. Zephyr; • cv. Sultan; • cv. Julia.

RAPID TEST FOR SUSCEPTIBILITY TO GASEOUS FLUORIDES

279

susceptible. There were parallel differences in the fluoride content of the leaf tips (Fig. 9): Zephyr was damaged with concentrations as low as 250/~gF/g, whereas Sultan and Julia showed damage only above 500 and 1750 ktgF/g, respectively.

70-

] 10

100 1000 Fluoride in leaf tips, ug/g

10000

Fig. 9. Percent leaf area of three varieties of Hordeum vulgare damaged by sodium fluoride solutions plotted against the fluoride content (pgF/g) of the leaf tips (0--5 cm): • cv. Zephyr; II cv. Sultan; • cv. Julia.

Horticultural species: The susceptibility of many horticultural plants, particularly the decorative monocotyledons like Gladiolus, is well known (e.g. Guderian et al., 1969; Spierings, 1969; NAS, 1971), so a number of common species were collected from rural gardens and tested. The species used and the minimum concentrations of fluoride needed to cause damage are shown in Table 3. Generally, the results were in agreement with the lists of relative susceptibility referred to above but there were some features of the experiment which need further comment. For example only Gladiolus and Crocus developed well-defined areas with the typical watersoaked appearance and were the only two species to develop dried, necrotic areas. The only symptom developed by most of the others was wilting (often severe) of the leaf tips followed by a gradual desiccation. The leaves of Endymion wilted in all of the solutions shortly after cutting and then slowly regained turgor over about 24 h. It is not known how this affected the experiment but as wilting undoubtedly affected fluoride uptake and translocation little reliance can be placed on the results.

280

A. W. DAV1SON, A. MARSLAND, W. E. BETTS

TABLE 3 COMI~ARISONOF TIIE REACTION OF SPECIES AND VARIETIESTO FLUORIDESOLUTIONS. MINIMUM CONCENTRATIONOF FLUORIDE(/ag/ml) NEEDED IN BATHING SOLUTIONTO PRODUCE PRONOUNCED LEAF DAMAGE

Species

Minimum concentration (ltgF/ml)

Gladiolus cv. Snow Princess Crocus ( u n k n o w n cultivar) Tulipa ( u n k n o w n cultivar) Narcissus poeticus cultivar Muscari ( u n k n o w n cultivar) Chionodoxa ( u n k n o w n cultivar) Galanthus nivalis Narcissus cv. King Alfred Endymion non-scriptus

1 <5 <5 5 10 10 25 50 >100

DISCUSSION

A detailed account of the entry of gaseous fluorides into leaves and of the production of visible symptoms is given by Treshow & Pack (1970). Most fluoride is thought to enter through the stomata, after which it penetrates the intercellular spaces and cells. It moves in the transpiration stream to the tips and margins where it accumulates. Analysis of sections (several millimetres long) of linear-leaved species usualty shows concentrations in the tips at least several times those in the basal regions (e.g. Zimmerman & Hitchcock, 1956). In many species the first visible sign of damage is the appearance of dark 'water-soaked' areas which are most easily seen by transmitted light. These then usually turn brown and desiccate within a few hours or days, depending on the weather. Some species react differently, however, to produce symptoms such as chlorosis (Spierings, 1969; Treshow & Pack, 1970). The response of cut leaves to fluoride solutions was generally similar to the response of gaseous HF. All of the linear-leaved species showed marked gradients from tip to base while the species with compound leaves showed differences from leaflet to leaflet. Fluoride solutions produced a water-soaked appearance and brown necrosis in many species but desiccation was very rapid and the affected areas sometimes remained predominantly green rather than turning brown. Presumably desiccation occurred so rapidly in the high temperature and low humidity of the growth cabinet that chlorophyll was not completely degraded. As with gaseous fluorides (Treshow & Pack, 1970) alteration of the physical environment around the leaf altered the pattern of fluoride accumulation and the development of visible symptoms. Leaf age is known to affect susceptibility to gaseous fluorides but the differences vary from species to species. In Pinus, the best known example (Treshow & Pack, 1970), the younger leaves are much more susceptible than older or mature leaves but in other species the reverse may be the case (Zimmerman & Hitchcock, 1956). In the present experiments the response of Zea rnais suggested that the older

RAPID TEST FOR SUSCEPTIBILITY TO GASEOUS FLUORIDES

281

leaves were more susceptible than younger leaves. There was certainly a difference in fluoride uptake and concentration with leaf age. Because climate, the physiological condition of the plant and genetic make-up all affect the response to gaseous fluorides, lists of the relative susceptibility of species and cultivars produced by different workers (e.g. Bolay & Bovay, 1965; Bossavy, 1965; Guderian et al., 1969; D~issler et al., 1972) often show variation in the ranking of the species. Thus it is not easy to compare in any detail the results of the present cut-leaf experiments with work on gaseous fluorides. However, the results were broadly consistent with published work; species such as Iris germanica, Zea mais, Gladiolus (cv. Snow Princess), Crocus purpureus (hybrid) and Narcissus poeticus were all visibly damaged at low concentrations, whereas Ligustrum vulgare, Triticum aestivum, Hordeum vulgare, Galanthus nivalis, Sambucus nigra and others were resistant to much higher concentrations of fluoride. This fact, coupled with the similarity in the pattern of concentrations and the symptoms produced, would suggest that the test is a valid, if crude, measure of the susceptibility of plants to gaseous fluoride. It is suggested that it could be employed for preliminary screening purposes or perhaps for investigation of the physiology or pathology of acute fluoride damage. The main practical problems encountered in using cut leaves and fluoride solutions were obtaining suitable material, holding leaves in the solution, wilting, senescence and variance within treatments. Obtaining suitable material was more difficult than was initially envisaged. Ideally all of the material should have been grown in the same condition but leaves taken from plants growing in the vicinity of the Newcastle University or any of the surrounding villages exhibited consistently high background fluoride concentrations because of emission from domestic fires (Davison et al., 1973), power stations and brickworks. Small or herbaceous plants could easily be grown in growth cabinets but shrubs and trees had to be collected from rural areas so the reactions of some species were not strictly comparable. Species such as Galanthus nivalis have reasonably large rigid leaves which were easily held with the base inserted in the solutions. Others presented problems. For instance, the smaller, less rigid leaves of Hordeum vulgare and Triticum aestivum tended to fold up and out of the solutions, and also to sag, thus allowing the upper part of the leaf to touch the solutions. Very small leaves such as Medicago sativa constantly became immersed in the solutions. A simple method of holding large numbers of leaves of different sizes would be an advantage. Several species wilted almost as soon as they were cut prior to experiment. Endymion has already been mentioned but Plantago lanceolata and Heracleum sphondyllium were also particularly prone to this problem. (As loss ofturgor almost certainly affected fluoride uptake and translocation, these species were not investigated further.) Cutting the leaves under water might prevent wilting, but it is virtually impossible with wild material.

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A.W. DAVISON, A. MARSLAND, W. E. BETTS

S o m e o f the cereals in p a r t i c u l a r were p r o n e to r a p i d senescence after cutting a n d possibly this c o u l d influence results even where the d u r a t i o n o f experiments was k e p t to the a b s o l u t e m i n i m u m required to p r o d u c e s y m p t o m s . T h e w i t h i n - t r e a t m e n t variance was generally high (see a c c o m p a n y i n g graphs for s t a n d a r d errors), a n d it was necessary to use at least l0 to 15 replicates per treatment. In certain cases (e.g. Hordeum vulgate) m o r e than 15 w o u l d have been better. The causes o f variance were p r o b a b l y differences in the age o r physiological state o f the leaves, slight variations in the physical e n v i r o n m e n t o f individual leaves, loss o f c o n t a c t with the solution a n d senescence. It should be possible to reduce this variance by i m p r o v e m e n t s in technique a n d s t a n d a r d i s a t i o n o f materials and methods.

ACKNOWLEDGEMENT The a u t h o r s wish to a c k n o w l e d g e the assistance o f the R o y a l Society.

REFERENCES BOLAY, A. & BOVAY,E. (1965). Observations sur la sensibilit6 gaz fluor6s de quelques esp&:es v6g6tales du valais. Phytopath. Z., 53, 289-98. BOSSAVY,J. (1965). Echelles de sensibilit6 au fluor. Revue for. ft., 17, 205-11. BORSDORF, W. (1960). Beitr/i.ge zur Fiuorsch~dendiagnostik. I. Fluorsch~iden-Weiserpflanzen in der Wildflora. Phytopath. Z., 38, 309-15. D.~SSLER, H. G., RANFT, H. & REHN, K. H. (1972). Zur Widerstandsf~ihigkeit von Geh61zen gegenfiber Fluorverbindungen und Schwefeldioxid. Flora, Jena, 161, 289-302. COOKE, J. A. (1972). Fluorine compounds in plants: their occurrence, distribution and effects. Ph.D. thesis, University of Newcastle upon Tyne. DAVISON, A. W., RAND, A. W. & BETTS, W. D. (1973). Measurement of atmospheric fluoride concentrations in urban areas. Environ. Pollut., 5, 23-33. GUDERIAN, R. H., VAN HAUT, H. d~. STRATMAN,H. (1969). Experimentelle Unterschungen fiber pflanzensch~idigende Fluorwasserstoff-Konzentrationen. ForschBer. Landes NRhein-Westf, No. 2017, 55 pp. NAS (1971). Biological effects of atmospheric pollutants: fluorides. Washington, National Academy of Sciences. SPIERINGS,F. H. F. G. (1969). A special type of leaf injury caused by hydrogen fluoride fumigation of narcissus and nerine. In Proceedings of the European Congress on the Influence of Air Pollution on Plants and Animals, 1st, 87-9. Wageningen, Centre for Agricultural Publishing and Documentation. TRESHOW,M. & PACK,M. R. (1970). Fluoride. In Recognition of air pollution injury to vegetation: A pictorial atlas, ed. by J. S. Jacobsen & A. C. Hill, D1-D17. Informative Report 1, TR-7 Agricultural Committee, Pittsburg, Air Pollution Control Association. ZIMMERMAN,P. W. t~ HITCHCOCK,A. E. (1956). Susceptibility of plants to hydrofluoric acid and sulphur dioxide gases. Contr. Boyce Thompson Inst. PI. Res., 18, 263-79.