New Phytol. (1997), 135, 501-515

Epicuticular wax of subarctic Scots pineneedles: response to sulphur and heavymetal deposition

BY M. T U R U N E N ^ * , S. H U T T U N E N ^ , K. E. PERCY^c. K. MCLAUGHLIN^ AND J. LAMPPU^

^Arctic Centre, University of Lapland, P.O. Box 122, FIN-96101 Rovaniemi, Finland^Department of Biology/Botany, University of Oulu, FIN-90570 Oulu, Finland^ Natural Resources Canada, Canadian Forest Service - Atlantic Forestry Centre,P.O. Box 4000, Fredericton, N.B. Canada E3B 5P7

{Received 11 April 1996; accepted 15 October 1996)

SUMMARY

The response of epicuticular wax of Scots pine {Pinus sylvestris L.) needles to dry- and wet- deposited sulphur andheavy metals was investigated at six sites located 10-110 km from the Monchegorsk Cu-Ni smelter on the KolaPeninsula, North-West Russia, and in a long-term irrigation experiment where pines were exposed over fourgrowing seasons (1991-1994) to either acid rain treatment at pH 3-1 (HgSO^), metal treatment at pH 5-7 (Cu andNi) or a combination of these at pH 3-1. Needle wettability exhibited a closer relationship with epistomatal waxtube distribution (WTD) than with chemical composition of epicuticular wax. Water droplet contact angles(DCA) decreased towards the smelter, and significant differences due to site were noted for 26-month-old and 38-month-old needles. Significant differences due to site were determined for secondary alcohols, dehydroabietic acidand hydroxy fatty acids, the proportions of which ranged from 22-5 to 48-9%, 6-2 to 22-4% and 0-6 to 2-6%respectively, depending on site and needle age class. The proportion of dehydroabietic acid increased towards thesmelter, but no gradient was observed in the proportion of secondary alcohols or hydroxy fatty acids. No majoreffect of experimentally applied pollutants on the chemical composition or structure of the epicuticular wax wasobserved. The effect of treatment on DCA was significant in 1993 and 1994 due to a 6-5-13-2 degree greaterwettability of the 37^9-month-old acid-treated needles relative to the irrigated or dry controls. Sulphuric acid atpH 3-1 did not increase needle wettability when combined with copper and nickel sulphate in similarconcentrations. These data indicate that S deposition, especially H^SO^, plays a more important role in needlesurface deterioration than Cu and Ni. Pollutant-induced changes in epicuticular wax structure and needlewettability mimic natural wax ageing, but at an accelerated rate. Changes in wax chemical composition might alsobe caused by pollutant-induced metabolic changes in elongating needles.

Key words: Pinus sylvestris (Scots pine), cuticle, epicuticular wax, wettability, sulphur and heavy metaldeposition.

and contamination of soil and ground water has beenINTRODUCTION reported near smelters in Sudbury, Canada (Freed-

The dry deposition of sulphur and heavy metal man & Hutchinson, 1980), as well as Nikel andcompounds, principally SOg and insoluble metal Monchegorsk, North-West Russia (Kozlov,particles, often constitutes the principal pathway of Haukioja & Yarmishko, 1993; Tikkanen & Niemela,pollutant transfer in areas close to the smelters due to 1995). Stressed conifers growing in the vicinity ofshort distances for dispersion and low residence smelters are often characterized by needle dis-times (Laurila, Tuovinen & Lattila, 1991; Tuovinen coloration, premature needle loss and inhibited shootei a/., 1993). Large-scale damage to forest ecosystems growth (Kozlov et al., 1993; Tikkanen & Niemela,

1995). The individual effects of S and heavy metalpollution on forest trees are often hard to separate in

* To whom correspondence should be addressed at: Arctic the field. As the distance from the pollution SOUrceCentre, University of Lapland, P.O. BOX 122, FIN-96101 increases, tree responses to low levels of fluctuatingRovaniemi, Finland. , , . r. i j u • i j

E-mail: mturunen@levi.urova.fi pollutant mixtures are often masked by spatial and

502 M. Turunen and others

temporal variation in soil and climatic charac- & Huttunen, 1988; Havas & Hyvarinen, 1990;teristics, fungal diseases and insect attacks (Kozlov e Staszewski, Godzik & Poborski, 1994; Turunen etal., 1993; Turunen & Huttunen, 1996). al., 1994).

It is postulated that air pollutants can alter the The wettability of needle surfaces is pre-structure and chemical composition of conifer needle dominantly determined by epicuticular wax struc-surfaces directly or indirectly, e.g. by modifying wax ture and chemical composition. The ratio of tubularbiosynthesis in developing needles, or by chemical to amorphous wax deposits and the distribution ofinteraction with needle waxes, in the case of functional groups in the wax molecules are factorsextremely strong acids, in situ (Percy, McQuattie & that play an important role in determining needleRebbeck, 1994). Direct chemical reactions between wettability since w-alkanes and alkyl esters, forneedle epicuticular wax and pollutants are, however, instance, increase hydrophobicity whereas hydroxyunlikely, because the chemically inert/stable charac- fatty acids reduce it (Holloway, 1970; Jagels, 1994).teristics of needle wax result in a lack of potential In addition, particle deposition and phyllospherereaction sites (Riederer, 1989). Moreover, impacts of micro-organisms might affect the wettability of thepollutants on epicuticular waxes vary according to surface, depending on their hydrophilic- hydro-the pollutant dose, genetics (species, clone), the phobic nature and the degree of micro-roughnessdevelopmental stage of the foliage, and environ- (Percy et al., 1993; Cape, 1994; Jagels, 1994; Stas-mental factors (Percy & Baker, 1990; Turunen & zewski et al., 1994; Schreiber, 1996). Since it is theHuttunen, 1990; Cape, 1994; Huttunen, 1994). contact time and area of water droplets and films on

The principal pathway for SOg uptake is through the surface of the foliage and cuticular propertiesthe stomata, whereas components of acid rain and that determine rates of ion and water transportwet-deposited Cu and Ni cations are principally across the cuticle, alterations in wetting charac-taken up from the soil (Wotton, Jones & Phillips, teristics of needle surfaces might be crucial for long-1986; Vesala et al., 1995). Field investigations in lived forest trees (see Haines, Jernstedt & Neufeld,SOg-polluted areas and experimental exposures to 1985; Tyree, 1994).acid rain/mist/fog at pH 3—4 have revealed damaged The aim of this investigation was to study theepicuticular wax structures and increased wettability effects of sulphur and heavy metal deposition on theof the needle surfaces in many conifer species (Cape, physico-chemical characteristics of subarctic Scots1983; Huttunen & Laine, 1983; Crossley & Fowler, pine needle surfaces by comparing observations1986; Turunen & Huttunen, 1990; 1991; Huttunen, made in the field with those received from a multi-1994; Jagels, 1994 and references therein). Sulphur year experimental exposure. The micromorpho-dioxide and acid rain/fog/mist have been found to logical and chemical responses of the epicuticularbe more damaging than O3 (Schmitt, Ruetze & wax and the wettability of the needle surface,Liese, 1987; Cape, Sheppard & Binnie, 1995), and together with pollutant accumulation in the needles,acid mist containing S more damaging to the needle were investigated in relation to distance from thesurfaces of Norway spruce and silver fir {Abies alba Cu-Ni-smelter emitting SO2 and heavy metals, andMill.) than that containing N (Rinallo et al., 1986; after two and a half, and/or four growing seasons ofCape, 1994). However, the responses of the needle treatment with simulated acid rain (H2SO4, pH 3),surfaces of blue spruce {Picea pungens 'glauca' heavy metals (Cu, Ni), singly or in combination.Engelm.) and jack pine {Pinus banksiana Lamb.) toSO, were negligible (Riding & Percy, 1985; Patrie &r, ' if^r^A^ J 1 - MATERIALS AND METHODS

Berg, 1994), and there are contrasting reports onNorway spruce (Cape, 1994; Cape et al., 1995) and Q- J • •Scots pine (Cape, 1983, Cape, Paterson & Wolfenden1989; Turunen et al., 1994). Five Scots pine {Pinus sylvestris L.) trees were

Insoluble and soluble heavy metal particles might sampled at six sites located in a transect extendingaccumulate on aerial surfaces of forest trees, and 10-110 km from the Monchegorsk smelter area onsoils, and might even penetrate bark. Only a minor the Kola Peninsula, North-West Russia (Table 1,proportion of heavy metal cations, however, is taken Tikkanen & Niemela, 1995). The sites comprisedup via stomata and cuticle (Little, 1973; Krause dry heath forest dominated by 60-236-yr-old nat-& Kaiser, 1977; Zottl, 1985; Tyree, 1994). The urally regenerated Scots pine. The locations of theresponses of conifer needles to wet-deposited heavy sites within forest damage zones with their modelledmetals are less well known, but wind-driven heavy SO2 concentrations (Tuovinen et al., 1993) aremetal particles are reported to erode epicuticular presented in Figure 1. The main sources of industrialwaxes, occlude epistomatal chambers and inhibit pollution in this area are the Cu-Ni smelters atstomatal closing. These effects have been linked with Monchegorsk which emit SO2 and heavy metals,detrimental changes in needle water relations and open-cast apatite mines and fertilizer factory atalterations in needle wettability characteristics Apatity, Fe, apatite and mica mines at Kovdor, and(Huttunen, Havas & Laine, 1981; Mankovska, Peura an Al smelter at Kandalaksha (Kryuchkov, 1993).

Pollution effects on cuticles of pine needles 503

Arctic Ocean30

Kevo

Koia PeninS\*laKandalaksha! Arctic Circle

Figure 1. The area studied, with sampling sites (•) and emission sources (® ) within different forest damagezones. I = forest death area (> 15 nl SOg 1~ ), II = inner visible damage zone (6-15 nl SOg 1~ ), III = outervisible damage zone (8-15 nl SOj 1 ), IV = inner non-visible damage zone (2-3 nl SOg 1" ) and V = outer non-visible damage zone (1-2 nl SO2 1" ) (modified from Tikkanen & Niemela, 1995). The annual wind rose (meanfrequency of wind direction for 1970-1990) is based on the data of the weather station at Monchegorsk (Berlin,1992).

Atmospheric SOg concentrations close to thesmelters are typically 15-20 nl SOg 1" (nl 1" = ppb)with 100-fold higher short-term peak concentrations(Tuovinen et al, 1993 ; Tikkanen & Niemela, 1995).The area falls mainly in the northern borealconiferous forest zones (Kalela, 1961). The tem-perature sum (day degrees, dd > + 5 °C) varies be-tween 678 and 967 dd, depending on the site, andannual precipitation is between 524 and 535 mm,about 30-40% of which is snow (Table 1). Theeastern sites are characterized by higher temperaturesums than those in the west. Further details arepresented elsewhere (Kryuchkov, 1993; Tuovinen etal, 1993; Luzin, Pretes & Vasiliev, 1994; Makinen,1994; Tikkanen & Niemela, 1995; Turunen, 1996).

The acid rain-heavy metal experiment was carriedout in the extreme north of Finland (69° 45' N,27° o r F) near the Kevo Subarctic Research Stationof the University of Turku (Fig. 1). The area (107 mabove sea level) lies within the mountain birch forestzone, but still has some isolated pine stands in theriver valleys. The experimental forest is located in adry heath type forest with Scots pines and mountainbirch {Betula pubescens ssp. tortuosa (Ledeb.)Nyman) the dominant tree species. The mean annualtemperature is — 2-0 °C and the length of the growingseason 110-125 d, minimum winter temperaturesoften falling below — 40 °C. During the experimentalperiod (1991-1994), the temperature sum variedfrom 517 to 637 dd and annual precipitation from366 to 578 mm. Mean annual SOg concentrationswere in the range 0-2-0-5 nl 1"^ and annual mean

ambient rain acidity was pH 4-8 (Kallio, 1975 ; FMI1991-1994a, b; Helander, 1994; Turunen, 1996).

Experimental design

The acid rain-heavy metal experiment consisted of25 plots (6 X 8 m) each containing at least onenaturally growing pine tree and one birch (Helander,1994). The pines were 30-50 years old and 1-8-5-3 mhigh, with diameter at 1 m of 1 •6-8-2 cm. A factorialdesign with randomized complete blocks wasemployed. Plots were grouped in sets of five to givea total of five blocks. The plots within each blockwere randomly assigned to the following treatments:lake water at pH 5-9 (irrigated control or IC),simulated acid rain treatment at pH 3-1 (A3), metaltreatment with Cu and Ni at pH 5-7 (M); acombination of acid and metal treatments at pH 3-1(A3*M) and un watered plots receiving only ambientrain, which served as untreated dry controls (DC).

The plots received ambient rainfall and simulatedrain (60% increase during the summer), which wassprayed on the foliage and soil in 5 mm amountstwice weekly with Hozelock® sprinklers. Dropletdiameters, measured using water-sensitive paper(Teejet Spraying Systems, Illinois, USA), rangedfrom 109 to 340/^m, with a plot-specific mean of243 /im. Application of simulated rain occurredduring the following periods: 20 July-27 August1991, 11 June-28 August 1992, 14 June-27 August1993 and 28 June-31 August 1994. The current-yearneedles were almost fully elongated when the

504 M. Turunen and others

Table 1. Description of the sites

Variable

Distance from the smelter (km)Altitude (m.a.s.l.)Temperature sum (dd, t > +5 °C)Mean temperature (Jan. °C)Mean temperature (July °C)Precipitation sum (mm)Stand properties

Tree age (yr)Tree height (dm)Diameter (mm at 1-3 m)Number of stems per hectareVolume (m )

Site

Kovdor

110280678

- 1 513

529

80135194408118

Jena

83160747

- 1 513

535

179114153878126

Upoloksha

63140783

- 1 413

530

20610314573388

Pirenga

48150111

-1413

530

806069

220036

Imandra

36240929

- 1 314

530

236109190400

95

Monchegorsk

10230915

- 1 414

524

606267

123321

treatments began in 1991. In 1992-1994 needleswere either emerging or elongating when the treat-ments began (Table 2).

The simulated rain for the A3 treatments wasmade by adding H2SO4 to water from LakeKevojarvi to give pH 3, and that for the Mtreatments by adding sulphate salts (Cu asCUSO4. 5H2O and Ni as NiSO^. 6H2O). In 1992, thelake water was filtered through an ion-exchangeresin column (AHSL filtering system, VartiainenEngineering Ltd., Finland) to remove humic ma-terial and to soften it. The IC rain was slightlyacidified with H2SO4 to pH 5 in order to approximateambient rain. The annual total deposition of S atKevo is 350 mgm"^, but some 200 km away, itexceeds 2000 mg m~ , as a consequence of thesmelter at Nikel on the Kola Peninsula. The fourgrowing seasons of cumulative S deposition appliedin this simulation experiment amounted to approxi-mately five times the ambient deposition at Kevo(Table 2, Laurila et al., 1991).

Sampling and measurements

Sampling. In the field study, Scots pine shoots (5-10per tree) were collected in August 1993 from themiddle third of the canopy (6—10 m). Five trees weresampled at each of six sites {n = 30). The twoyoungest needle age classes (2-month-old and 14-month-old needles) were later removed in thelaboratory for scanning electron microscopy (SFM)and determination of wax chemical composition andneedle element concentrations. All needle age classespresent (from 2—74-month-old needles) weresampled for wettability measurements.

In the acid rain—heavy metal experiment, shootsand needles were sampled on 19 July 1993 and 2October 1994 from the middle part of the canopytreated with simulated rain after two and a half andfour growing seasons of treatment, respectively. Thematerial sampled included needles elongated beforethe experiment started (1987-1990), and those

elongated during the experimental period (1991-1994), representing needle ages of 0-5-73 months.Fach treatment was replicated five times for all thevariables investigated.

Epicuticular wax structure. Two to three needles perage class per tree (4—12 needles each tree, site,treatment) were examined by SFM. Needles wereair-dried at room temperature and a 10 mm segmentwas cut from the middle of each, mounted on stubswith double-sided tape and sputter-coated with gold-palladium (80:20%, 45 nm) using a Polaron 'Cool'Sputtering System (Type F5100, no 3DF51OO,Polaron Fquipment Ltd. London) equipped with amagnetic shielding target and specimen stage cooledwith a thermoelectric 'Peltier' device. Abaxialsurfaces were examined under a SFM (JFOL JSM-6400, no. 3D 6400, Jeol Ltd., Tokyo) operated at12 kV. Fach needle sample was scored in SFMmicrograph areas of 0'061 mm^ as follows: class 0,epistomatal wax tube distribution 100 % (poorlydeveloped, but well preserved); class I, 100% (welldeveloped and preserved); class II, 71-100%(slightly eroded); class III, 31-70% (moderately-eroded) ; class IV, 1—30 % (severely eroded); andclass V, 0% (very severely eroded) (Turunen &Huttunen 1991). The condition of the waxes in 2—4middle stomatal rows on the abaxial side of eachneedle were assessed in a random order.

Wax chemical composition. The needle age classeswere separated (5—10 branches each tree, site,treatment) and stored in a freezer ( —20 °C for 1 wk).Later, 5 g (f. wt) of needles were immersed in HPLCgrade chloroform (CHCI3) for 15 s and thechloroform/wax solution filtered through CHCI3-washed filter paper (pore size 1-S ptra). Followingpartial solvent evaporation, solutions were trans-ferred onto tared aluminium containers and filterpapers dried in a desiccator. Surface accumulation ofparticulate was determined by weighing ( + 0-0001 g)the filter papers. Wax samples were subsequently

Pollution effects on cuticles of pine needles 505

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redissolved in CHCI3, reduced to 500/^1 using arotary evaporator and transferred to preweighed( + 10/ig) glass ampoules. Solvent was removedunder a constant stream of N at 30 °C and the vialsre weighed to determine wax quantity.

Wax samples were prepared for chemical analysisusing column chromatography techniques modifiedfrom Percy & Baker (1990). The stationary phasewas silica gel, 200-400 mesh, 60 A (Aldrich,Milwaukee, USA). The column was eluted withsuccessive aliquots of hexane, hexane/chloroform(2/1 (v/v)), chloroform/ether (2/1 (v/v)) and meth-anol. The first three fractions were combined forquantitative analysis of chemical composition using aVarian 3410 high-temperature gas chromatograph(GC) fitted with a flame ionization detector (FID).Retention times were determined on a J&W DB-1H T fused silica capillary column (15 m long;0-32 mm internal diameter) with method siliconeliquid-phase (0-25 [im film thickness) using a carriergas flow of 4-4 ml min~^ He at 70 °C. Columnprogramming was 70-120 °C at 20 °C min"^ and120-390 °C at 6 °C min^\ The septum-program-mable injector was operated at 75-125 °C at18°Cmin-^ 125-395 °C at 12°Cmin-^ hold15 min. The FID was programmed isothermally at400 °C. The wax samples were silylated with N,O-bis (trimethylsilyl) acetamide (BSA) at 50 °C for30 min and 1 fig analite was injected onto thecolumn. A Varian Workstar (version 4.01) softwaresystem was used to integrate peak areas and calculatepercentage homologue composition ( + 0-0001%).Relative retention times (RRT) were calculated fromthe co-injection of a cholesteryl «-octanoate. GCassignments were made on the basis of appropriatewax homologues. Confirmation of GC assignmentswas made by gas chromatography-mass spectro-metry (GC-MS) as described previously (Percy &Baker, 1990).

Epicuticular wax chemical composition is pre-sented as proportion by homologue identified in eachwax sample recovered. Given the range in edaphicand phenoiogical characteristics across the studysites, expression of wax composition as a function ofneedle oven-dry weight was not utilized here.Duration of wax extraction in CHCI3 was identicalfor needles collected from both field and exper-imental sites.

Needle surface wettability. Needle wettability wasdetermined by static water droplet contact anglemeasurement (DCA) (Cape, 1983) performed onneedle material stored in a freezer. Measurementswere made on thawed needles after 10-15 min atroom temperature. A 1 /il water droplet was placedon the needle surface and the angle between theneedle surface and the tangent of the droplet wasmeasured under a light microscope with a protractorgraticule. The data were expressed as means of

506 M. Turunen and others

adaxial and abaxial surfaces (2—5 needles each ageclass, tree, site, treatment).

Elemental analysis. Concentrations of S, Ni, Cu, Fe,Ca and P in unwashed needles from five trees per sitewere analysed on a sequential X-ray spectrometer(Siemens SRS 3O3AS, no. 3D 3O3AS, Siemens Ag,Karlsruhe) equipped with a Siemens AG Rh 66 Rhanode, a LiF 100 (lithium fiuoride) crystal for Ni,Cu, Fe and Ca and a P F T (pentaerythritol) crystalfor S and P. In the acid rain-heavy metal experiment,S, Ni and Cu concentrations were measured fromthe chloroform-extracted needles from five trees pertreatment.

The needle age classes were separated and oven-dried at 60 °C for 48 h. Needles were homogenizedto a fine powder in a SAKO mill (KoneteollisuusLtd, type 120, 2800 r.p.m.) and pressed into pellets(200 kN). The following commercial standard ref-erence materials were used: SRM 1575, SRM 1572,CRM 061 and CRM 062. Control pellets wereobtained by adding known concentrations of

)2SO4, NiCla, CuCla, FeClg, NH.H^PO^ andg. 2H.2O.

Statistical analysis. A continuous variable for class ofwax structure was calculated from the estimatedcategorial variable using the following median valuesfor the classes: 0 + 1 , 100 % ; II, 85 % ; III , 50 % ; IV,15 %; and V, 0%. A value for the epistomatal waxtube distribution (WTD, %) of each tree and needleage class was calculated as described elsewhere(Turunen, 1996; Turunen & Huttunen, 1996).

Data were log-transformed in the case of Ni andCu concentrations in unwashed needles to normalizethe distributions. In addition to descriptive statistics,two-way ANOVA (Type III SS) was used to test maineffects of site or treatment, needle age, and theirinteraction, the unbalanced nature of the experimentbeing taken into consideration where necessary(various numbers of needle age classes investigated).Within each needle age class, the site/treatmenteffects were additionally tested by one-way ANOVA,

pairwise comparisons being made using Tukey'sStudentised Range ^-test (HSD). The variationamong the trees within each site/treatment was usedas an error term.

The relationship between the needle surfacevariables and needle element concentrations wasstudied by means of Spearman's rank correlationcoefficients {rJ using tree-specific {n = 25 for theacid rain-heavy metal experiment, and « = 30 for thefield study) and site-specific (n = 6) mean values.Site-specific mean values were always used whenstudying the effect of atmospheric SOg concen-trations, which were extrapolated spatially for eachsite (Tuovinen et al., 1993), and of distance from thesmelter on the variables investigated.

RESULTS

Pollutant accumulation and visible damage

Particle accumulation in wax recovered from needlesurfaces ranged from 5-7 to 39-0 x 10"^ g (g"^ f. wt)and was greater on older needles (Tables 3,4). In thevicinity of the smelters, SFM and FDS indicatedthat particles containing S, Ni, Cu and Fe werescattered over the whole needle surface, but hadaccumulated to greater extent in the epistomatalchambers, particularly in older needles. Needlesurface accumulation of Ni and Cu was measurableon 14-month-old needles at distances up to 63 and48 km respectively from the Monchegorsk smelter,amounting to over 40 % of the total concentration ofthese elements in the needles at a distance of 10 kmand 20 % at 36 km (unpublished). Concentrations ofS, Ni, Cu, Fe and P in the 2-month-old and 14-month-old needles collected in August 1993 weregreater closer to the smelter and were significantlydifferent between sites. Ranges were: S 769-1269 ppm, Ni 2-154 ppm, Cu 2-77 ppm, Fe 23-195 ppm, P 853-1671 ppm and Ca 1434-4109 ppm,depending on the site and needle age class (Table 3).

In the acid rain—heavy metal experiment, needlesurface accumulation of particles was not related totreatment, but the effect of treatment on S, Ni andCu concentrations in the wax-extracted needles wasgreater than that of needle age (Table 4.). The A3*Mand A3 treatments increased S concentration by21-22%, and the M treatment increased the Niconcentration by 59%. Cu concentrations wereincreased by the A3*M treatment, and were larger inolder needles. The Cu concentration in the 13-month-old A3*M-treated needles was greater thanthat in the untreated DC by (89 %) or A3 by (75 %)treated needles, and that in the 25-month-old A3*Mneedles was greater than that in the DC needles by(73%).

Pines growing at sites 8—36 km from theMonchegorsk smelter retained only 3-yr-old to 4-yr-old needles whereas those further away retained 5-6needle year age classes. Two-month-old pine needleswere visibly undamaged at all sites. Needle dis-coloration on older needles included chlorosis andnecrosis of needle tips and was more evident close tothe smelters and increased with needle year age. Inthe acid rain—heavy metal experiment, the IC-treatedtrees had a mean of 5-8, and the A3*M- treated treesa mean of 6-6 needle age classes. Differences betweentreatments were not significant.

Epicuticular wax structure

Significant differences were determined relative toneedle age and site/treatment in epistomatal waxtube distribution (WTD) although variation amongtrees at each site/treatment and within each needle

Pollution effects on cuticles of pine needles 507

Table 3. Site-specific total concentrations of the pollutants S, Ni, Cu, Ee, P, Ca and P and particle depositionas determined from the wax extract at different distances from the smelter

Variable

Particles(10'^ g f. wt)

S (ppm)

InNi (ppm)

InCu (ppm)

Fe (ppm)

P (ppm)

Ca (ppm)

Agef

2142

142

142

142

142

142

14

F(site)

*

****************************#**n.s.n.s.

Distance i

110

9 428-1 abc

802 a783 a

4 a2 a3 a2 a

37 a71 ab

1284 a853

18323543

romi the smielter (km)

83

5-7: 17-2 C

849 ab769 a

7 b4 b3 a2 a b

23 a47 a

1317a928

1434 a2403

63

6-723-5 be

859 ab782 a

8 be5 b4 a b3 b

29 a74 ab

1369 a873

20483311

48

14-30-

966920

11146

113684

1572103321053773

27 a bababcdcbdabc

36

7-122-7 be

1031 be965 b

12cl17e12c17d33 a90 b

1671 be10382597 b4109

10

14-139 a

1177e1269 c

46 e154 d25 d77 e67 b

195 c1665 c101520403564

r^ (distance)

-0-4286 n.s.-0-4286 n.s.-1-000***-0-8286*-1-000***-1-000***-0-9856***-0-9856***-0-3714 n.s.-0-9429**-0-9429**-0-7714 n.s.-0-6000 n.s.-0-6571 n.s.

The effeet of site within each needle age class is tested with one-way ANOVA (statistical significances: *P < 0-05,**P < 0-01, ***P < 0-001), and pairwise comparisons with Tukey's Studentised HSD-test. Means marked with dif-ferent letters differ significantly at the P < 0-05 level. The relationship with distance from the smelter was studiedby Spearman's rank correlation co-efficient (r ,). n = S trees.

f Needle age (months).

Table 4. Particle deposition and S, Ni and Cu concentrations {with SD) in wax-extracted pine needles aftertreatment for two and a half growing seasons in July 1993

Variable

Particledeposition(10-5 g £ , -)

S (ppm)

Ni (ppm)

Cu (ppm)

Needle age(months)

1325

132513251325

F

n.s.n.s.

*o********o

Treatment

D C

6-0 (2-8)13-0(3-8)

731 (166)697 (92)

2-0 (0-5) a2-0 (0-7) a1-0 (1-5) c3-0 (4-0) b

IC

7-9-

737619

2-1-5-7-

4 (6-8)2 (3-2)

(84)(127)0 (0-9)0 (0-5)0 (2-8)0 (3-4)

aaabab

A3

11-0(4-2)17-0(11-2)

891 (93)880(177)

1-0 (0-5) a1-0 (0-0) a4-0 (2-5) be7-0 (3-1) ab

M

49

707661

447

10

-9 (5-0)-7 (3-7)

(100)(86)

-0 (0-8)-0 (0-9)-0 (3-6)-0 (6-3)

bbabab

A3*M

10-0(5-1)16-0 (9-6)

933(1689)827(238)

2-0 (0-4) a2-0 (1-5) a9-0 (2-9) a

11-0 (0-4) a

The main effect of treatment within each needle age class is tested with 1-way ANOVA (statistical significances:oP < 0-1, *P < 0-05, **P < 0-01, ***P < 0-001) and the pairwise comparisons with Tukey's Studentised Range ^-test.Means marked with different letters differ significantly at the P < 0-05 level, n = 5 trees. Abbreviations: DC, dry control;IC, irrigated control; A3, acid rain pH 3; M, metal treatment (Cu and Ni); A3*M, combination of A3 and M.

class was relatively high (coefficient of variation30-40%). Decline in W T D due to ageing was mostpronounced after the first overwintering (Fig. 2a-c,Table 5). At Kevo, 47 % of wax tubes in epistomatalchambers observed at 0-5 months were no longerevident at 13 months. Wax tubes projecting highestabove the needle surface were the first affected.

Significant differences (P < 0-01) in W T D due tosite were noted for 14-month-old needles (Fig. 2 a).The W T D on the 14-month-old, unlike thaton the 2-month-old needles, was significantly cor-related with the accumulation of pollutants inthe needles as follows: Cu (r, = -07028***) ,Ni {r = .0-6771***), Fe (r, = -0-5748***), S

(y^=,_0-5382**) and P (r, =-0-3855*). The site-specific relationship between distance from thesmelter and atmospheric SOg concentration, andWTD was also significant {r^ = 0-8857* andf = —0-8857*, respectively).

In the acid rain-heavy metal experiment, declinein WTD due to needle age was significant {P < 0-05)in 1993 and 1994. The effect of treatment wassignificant in 1994 following four growing seasons oftreatment. This was due to a W T D in the 27-month-old needles of the M-treated 26 % lower than in thetrees as compared with the DC trees. Despite thefairly high WTD in A3 treatment, the wax tubeswere often short.

508 M. Turunen and others

100

ab a abc be abc c

100

13 25

15 27

Needle age (months)39

Figure 2. Epistomatal wax tube distribution (WTD, %)on the needles in August 1993 at different distances fromthe smelter (D HO km, M 83 km, g 63 km, 0 48 km, H36 km and • 10 km) (a), in acid rain-heavy metalexperiment in 1993 after treatment for two and a halfgrowing seasons {b), and in 1994 after four growing seasons(c), (O DC, m IC, g A3, H M and • A3*M,abbreviations as in Table 2). Each bar is the mean for fivetrees. Different letters above the columns indicate stat-istically significant differences between the sites/treatments within each needle age class at the P < 0-05level in Tukey's Studentised HSD test.

Wax chemical composition

The percentage of homologues resolved in epi-cuticular wax recovered from Scots pine needles.

identified by GC and confirmed by GC-MS, rangedfrom 80—85 %. Epicuticular wax recovered from 13-month-old needles at Kevo comprised 10 classes(Fig. 3). Secondary alcohols (40-2%), alkyl esters(12-9%), nonacosane diols (6-6%), fatty acids(6-2%), alkanes (3-9%), primary alcohols (2-4%),and hydroxy fatty acids (0-7 %) were resolved by GCand confirmed by GC-MS. Three diterpene acids,dehydroabietic (6-7%), pimaric (2-2%) and abietic(1-2%) were also confirmed by GC-MS. Estolides(13-3%) were resolved by GC and tentativelyidentified using RRTs previously calculated frominjection of red spruce wax (Percy, Jensen &McQuattie, 1992). Di-esters (3-8%) were resolvedby GC, but not confirmed.

The only secondary alcohol homologue confirmedwas nonacosan-10-ol (C29). Alkyl esters were prin-cipally even-chained homologues (C34—Cg4), but odd-chained homologues (C45-C49) were also confirmed.A homologous series of 10 even-chained fatty acids(Cj 4—C32) were identified, the dominant ones beinghexadecanoic acid, octadecanoic acid and octaco-sanoic acid. Four nonacosane-diol isomers ( — 3,10,— 4,10, —5,10 and —7,10) were confirmed, with— 4,10 dominant. Alkanes included both even-chained and odd-chained homologues in the rangeCi8~C3o. Primary alcohols confirmed included tetra-cosanol, pentacosanol, hexacosanol, octacosanol andhentriacontanol. Hydroxy fatty acids confirmedincluded hydroxy-dodecanoic acid and hydroxy-hexadecanoic acid.

The variation in wax chemical composition withinindividual sites/treatments and needle age classesdepended on the wax class, being highest for hydroxyfatty acids (coefficent of variation 75-100%) andlowest for secondary alcohols and fatty acids(coefficient of variation 15—20%). The 14-month-old needles collected from near the smelters hadsignificantly {P < 0-05) smaller proportions of sec-ondary alcohols, dehydroabietic acid and hydroxyfatty acids than 2- month-old needles, and higherproportions of alkyl esters, estolides and di-esters. Inthe acid rain—heavy metal experiment, ANOVA indi-cated significant differences {P < 0-05) in wax com-position between the needle age classes in all the waxclasses except for secondary alcohols and estolides.Proportions of diterpene acids (7-3%), alkanes(2-9%), di-esters (3-1%) and hydroxy fatty acids(0-2%) were smaller in the 25-month-old needlesthan in the 13-month-old needles, and the pro-portions of alkyl esters (15-6%), fatty acids (8-4%),nonacosane diols (8*2 %) and primary alcohols(3-2%) were higher.

In the vicinity of the smelters, significant effectsdependent on site were found for secondary alcohol,dehydroabietic acid and hydroxy fatty acid, theproportions of which ranged from 22-5 to 48-9 %, 6-2to 22-4% and 0-6 to 2-6 % respectively (Fig. 4, Table5). The proportion of dehydroabietic acid increased

Pollution effects on cuticles of pine needles 509

Table 5. Main effects of needle age, site/treatment and their interaction tested by ANOVA for the cuticularvariables and pollutant accumulation

Variable

Cuticular variableWTD

Secondary alcoholEstolideAlkyl esterDiterpene acid

Dehydroabietic acidPimaric acidAbietic acid

Nonacosane diolFatty acidAlkaneDiesterPrimary alcoholHydroxy fatty acidDCA

Pollutant accumulationParticle depositionStNifCut

Year

19931994199319931993

19931993199319931993199319931993199319931994

1993199319931993

Field

Age

***

********

***n.s.n.s.n.s.n.s.n.s.**#n.s.****

***

n.s.n.s.***

study

Site

*

*

n.s.n.s.

*n.s.n.s.n.s.n.s.n.s.n.s.n.s.****

#****#******

(Age X Site)

**

n.s.n.s.n.s.

n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.

n.s.n.s.******

Experiment

Age

******n.s.n.s.***

**************************

**

n.s.n.s.*

Treatment

n.s.***

n.s.n.s.n.s.

n.s.n.s.n.s.n.s.n.s.n.s.*n.s.n.s.******

n.s.********

(Age X Treatment)

n.s.n.s.n.s.n.s.n.s.

n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.

n.s.n.s.n.s.n.s.

WTD, wax tube distribution; DCA, droplet contact angles. Statistical significances: *P<0-05, **P<0-01,***P< 0-001.

t In the experiment, elemental concentrations were determined from wax-extracted needles.

50

40

co

oQ.EooX<0

30

20

10

Secondary alcohol

Estolide

Q ] Alkyl ester

Diterpene

^ Diol

{[J] Fatty acid

^ H Alkane

^ g Di-ester

{ ^ Primary alcohol

EB Hydroxy fatty acid

13 25Needle age (months)

Figure 3. Chemical composition (%) of the epicuticular waxes recovered from 13-month-old and 25-month-old needles of untreated DC pines in July 1993. M = 5 pines. Abbreviations as in Table 2.

significantly towards the smelter (r, =-0-8697*), In the acid rain-heavy metal experiment,whereas no gradient was observed for secondary significant changes in wax composition were detectedalcohols and hydroxy fatty acids. at the class level, but there was greater variation due

510 M. Turunen and others

Secondary alcohol (%)

n o 83 63 48 36 10km

Fatty acid (%)

110 83 63 48 36 10km

110 83 63 48 36 10km

Did (%

110 83 63 48 36 10km

20

15

10

5

0

Estolide (%) Di-ester (%)

(e)

AAAAM110 83 63 48 36 10

km110 83 63 48 36 10

km

Primary alcohol (%) Alkane (%)

110 83 63 48 36 10km

Hydroxy fatty acid (%)

110 83 63 48 36 10

km

110 83 63 48 36 10

km

Dehydroabietic acid (%)

110 83 63 48 36 10km

10

8

6 -

4 -

Abietic acid (%) PImaric acid (%)

ik)

110 83 63 48 36 10km

110 83 63 48 36 10km

Figure 4. Proportions (%) of {a) secondary alcohols, (b) alkyl esters, (c) fatty acids, {d) nonacosane diols, (e)estolides, (/) unconfirmed di-esters, {g) primary alcohols, {h) alkanes, {i) hydroxy fatty acids and diterpene acids,including 0) dehydroabietic, {k) abietic and (/) pimaric acid at different distances from the smelter in August1993. D 2- and B 14-month-old needles. Note the different scales. Each bar is the mean for five trees.

to needle age class than due to treatment (Table 5). proportions of nonacosan-10-ol and estolides wereThere was no (age x treatment) interaction. After not affected by needle age or treatment,two and a half growing seasons, the effect oftreatment on wax chemical composition was

.J, 1 r J- - r 1 - 1 Needle surface wettabilitysignificant only tor di-esters, proportions or whichwere the lowest in the M-treated (2-5-2-8%) and Theeffectsof needle age and site/treatment on waterhighest in the A3-treated samples (3-6-4-3 %). The droplet contact angles (DCA) were significant,

Pollution effects on cuticles of pine needles 511

100

90

80

70

60

50

100

(a)

-

IIIa ab c

ab ab be a

IML

a ab ba ab

11 lli

L .

I>

14 26 38 50 62

0-5 13 25 37 49 61 73

70

60

50

a ab b abab

I

15 27Time (months)

Figure 5. Water droplet contact angles (DCA, degrees) forall the existing needle age classes in August 1993 atdifferent distances from the smelter (D HO km, [U 83 km,^ 63 km, S 48 km, H 36 km and B I O km) (a), in acidrain-heavy metal experiment in 1993 after treatment fortwo and a half growing seasons {b), and in 1994 after fourgrowing seasons (c) (D DC, [U IC, ^ A3, 0 M and •A3*M, abbreviations as in Table 2.). The needles olderthan 25 months in 1993 had formed before the experimentstarted, but in 1994 all the needles had been formed duringthe experiment. Columns marked with different lettersdiffer significantly at the P < 0-05 level in Tukey'sStudentised Range i-test. n = S pines.

although no age x site interaction was observed (Fig.5 a, Table 5). The variation in DCAs (coefficient ofvariation 5 %) within each needle age class andsite/treatment was lower than for W T D or waxchemical composition. Decrease in DCA due toageing was most pronounced after the first over-wintering.

In the vicinity of the smelters, significantdifferences between sites were found among the 26-month-old and 38-month-old needles, but notamong the younger or older needles. Smallest angleswere measured on 38-month-old needles, the last ageclass retained, at a distance of 10 km from the

90

85

(D

< 75oQ

70

65

14-month-oldr^ = 0-9429P= 0-0048**

•

-

• •

needles

0

• •

2-month-oldTg = 0-2000P= 0-7040 n.

00

%

0

tneedles

s.

0 20 40 60WTD (%)

80 100

Figure 6. Relationship between WTD and DCA atdifferent distances from the smelter in August 1993. r^ =Spearman Rank correlation coefficient. Each plot is themean for five trees.

smelter, and the largest angles for 2-month-oldneedles at 48 km. The relationship between DCAand 'pollutant variables' was not significant forthe youngest needles, but for those that hadoverwintered it was stronger and increased withneedle age. The relationship between DCA andneedle pollutant concentrations in 14-month-oldneedles was as follows: P (r =—0-5725***), S(r =-0-5072**), Cu (r =-0-4923**) and Ni(fg = —0-4202*). Both the distance from the smelterand atmospheric SO2 concentration were related toDCA (r, = 0-9429** and r =-0-9429**, respect-ively). The DCA was also related to W T D , with treeand site relationships being significant for 14-month-old needles {r^ — 0-45676**, n = 30 and r^ =0-94286**, n — 6, respectively), but not for 2-month-old needles, which had not overwintered (Fig. 6).

In the acid rain-heavy metal experiment, theeffects of needle age and treatment were highlysignificant in 1993 and 1994 (Fig. 5 b, c Table 5). TheDC needles generally had higher DCAs than the IC-treated or those receiving the other treatments(although not significant). Significant effects of thetreatments on DCA after two and a half growingseasons were found only in the older needle ageclasses (P < 0-05 for 37-month-old needles and P <0-01 for 49-month-old needles), which had beenelongated 1—2 yr before the experiment started. Thiswas due to the A3 treatment having DCAs 11-8-13-2degrees lower than those of the DC or IC controls.The higher Cu concentration in the wax extractedfrom the 25-month-old needles (1-9 ppm) coincidedwith lower DCAs (r, = -0-49684, P < 0-05, n = 24),but no significant correlations were found for theaccumulation of S or Ni.

In 1994, significant {P < 0-05) treatment effects onDCA were observed in the youngest and oldestneedle age classes elongated during the course of theexperiment. The effects of the treatments wereattributed to lower DCAs on A3*M-treated 3-month-old needles (5°) and A3-treated 39-month-old needles (6-5°) as compared with the DC controls.

512 M. Turunen and others

but not with the IC ones. Lower DC As were explanation for changes in wax chemistry close to thesignificantly related to reduced WTDs in both the smelters indirectly, e.g. through pollutant-inducedyoungest (r = 0-73259, P < 0-001, n = 25) and nutrient deficiency. It has been shown that epi-oldest (/'g = 0-46702, P < 0-05, n = 23) needle age cuticular wax morphology of Scots pine seedlingsclasses. changed from tube-like structures to more fused and

net-like ones as a result of soil-mediated deficienciesin K, Mg and Ca concentrations (Ylimartimo et al.,

DISCUSSION . r ^ ^ / ^ T T , 1 • r ^ ^

1994). High peak concentrations of SO, are commonThe present results indicate structural and chemical in the vicinity of the smelters, especially during thealteration of epicuticular wax and increased growing season (Laurila ei a/., 1991; Tuovinen eif a/.,wettability of the Scots pine needle surfaces in a 1993) resulting in greater damage to substomatalrelation to their proximity to smelters in a subarctic mesophyll cells than to epidermal cells (Holopainenenvironment. The decrease in the epistomatal WTD, et al., 1992; Kukkola et al., 1994), the sites ofand the associated increase in needle wettability, epicuticular wax synthesis. Sulphur dioxide mightobserved here both in the field and after pollutant also react with aldehyde constituents of epicuticularapplications, are in agreement with earlier field wax if these were present but the most likelyinvestigations on Scots pine in SO.j-polluted areas mechanism for the effect of SO2 on the needle(Cape, 1983; 1986; Huttunen & Laine, 1983; surfaces is through its dissolution in the water filmCrossley & Fowler, 1986), but the response of covering the needle surface, or water droplets toepicuticular wax composition to SO. , and heavy form HgSO^ (Cape, 1994). In developing needles,metals remains poorly understood. which have more permeable cuticles and thinner,

Epicuticular wax of Scots pine needles was found less lignified, epidermal cell walls than overwinteredto be comprised of ten classes (up to 70 homologues) needles (Walles, Nyman & Alden, 1973 ; Esau, 1977;each differing in number and orientation of func- Huttunen, Turunen & Reinikainen, 1989), aqueoustional groups. Although recovery of most wax classes acidity might disturb pH-sensitive enzyme systemsresolved in this study following a 15 s needle involved in wax biosynthesis (Percy et al., 1994).immersion in CHClgWas likely to have been relatively Differences in water retention on needle surfacescomplete, this might not have been the case for the might, at least in part, explain confiicting effectspolymeric estolides. Here, diterpene acids made up observed in many earlier SO2 fumigation experi-15 % of the Scots pine needle epicuticular wax, and ments (Patrie & Berg, 1994; Cape et al., 1995).have been previously identified as constituents in In the acid rain—heavy metal experiment, theepicuticular waxes of Monterey pine {Pinus radiata) wettability response to pH 3 H2SO4 became visible(Franich, Wells & Holland, 1978), Norway spruce first in older, naturally 'weathered' needles which{Picea abies) and Sitka spruce {Picea sitchensis) had been formed before the experiment started,(Priigel, 1994), but never in Scots pine. indicating chronic direct acid rain effects. Sulphuric

The proportion of dehydroabietic acid in epi- acid has been reported to induce alkyl ester hy-cuticular waxes increased towards the smelter and drolysis, resulting in the liberation of fatty acids ondifferences due to site were also found for secondary waxy surfaces (Percy et al., 1994), but this might notalcohol and hydroxy fatty acid. The increase in the have been the dominant mechanism for the changesproportion of dehydroabietic acid might be a meta- observed here, as no significant differences betweenbolic response of Scots pine to SOg and heavy treatments and between sites were found for alkylmetals. It is also possible, however, that dehydro- esters. This is probably because wax esters are onlyabietic acid might be largely infiuenced by phenotype hydrolysed at extreme acidities (pH < 3). Such(Kainulainen, Holopainen & Oksanen, 1995), since conditions are probably restricted to localized areasthe subarctic Scots pines exposed to H2SO4, CuSO^ on the needle surface, e.g. as a result of repeatedand NiSO4 at pH 3 (4-4 g S m"^ 0-04 g Cu m^^ wetting-evaporation cycles (Milne, Crossley &0-02 g Ni m" ) for two and a half growing seasons did Henderson, 1990; Percy et al., 1994). Furthermore,not manifest significant changes in the proportion of ambient rain acidity in the Kola area is highlydehydroabietic acid. Kainulainen ei a/. (1995) found variable due to alkaline dust (e.g. Ca5recently that SO2 fumigation with 100-155 nl T^ for (F,C1,OH)(POJ3) emissions from the Apatity18-20 d reduced the concentrations of palustric and fertilizer factory and from the mining of apatite oresneoabietic acids in current-year needles of Scots pine at Kovdor (Kozlov et al., 1993).seedlings, whereas effects on Norway spruce were The present results show that the wettability ofclone-dependent. However, the most common overwintered 26-49-month-old needles, whichditerpene acids in pine needles, abietic and dehydro- increased towards the smelters and resulted fromabietic acids, were not affected by SO2 fumigation. pH 3 H2SO4 treatment was related more to a reduced

Both SO2 and heavy metals have been reported to epistomatal WTD than to altered epicuticular waxperturb a wide range of metabolic processes in forest chemical composition. Relationships with the pro-trees (Darrall, 1989), and this is the probable portion of nonacosan-10-ol which crystallizes into

Pollution effects on cuticles of pine needles 513

the tubes visible by SEM (Jeffree, Baker & Holloway, a result of interaction between H+ and the divalent1975), and which accounted for 40% of the total cations, which might have reduced H+ ion uptakemass of epicuticular wax were less clear. Previous (Smalley, Hauser & Berg, 1993).field observations on Scots pine have indicated a A relationship between ageing and pollutants hasrelationship between decreased amounts of long- been suggested for a long time (Huttunen & Laine,chained alcohols and ketones, damaged epicuticular 1983; Huttunen, 1994). In the present study,wax structure and increased wettability of the needle pollutant deposition resulted in accelerated rates ofsurface in an area experiencing 15 nl SOg \~^ (Cape, needle surface alteration, an effect associated with a1983; 1986; Crossley & Fowler, 1986), but effects on decline in WTD and increased wettability of thewax structure were not always related to altered needle surface. Site-dependent changes in thechemical composition (Giith & Frenzel, 1989 a, 6; chemical composition of the epicuticular wax did notSchreiber, 1996). However, our observations are exclusively follow the ageing pattern, and werepartly consistent with the theory of Riederer et al. possibly due to pollutant-induced metabolic changes(1994), who suggested that the erosion of epicuticular in the developing needles. This was most evident inwax structures induced by natural ageing or by the proportions of dehydroabietic acid, whichpollutants does not always involve chemical changes increased with decreasing distance from the smelter,but could represent a change from a thermo- No significant site-dependent differences were founddynamically labile tube-like form to a more stable for alkanes, representing the most hydrophobic waxplate-like form. Surface deposition of heavy metal constituent (Holloway, 1970), although a recentparticles (e.g. Fe^S^, iron pyrites; CuFeS.2, copper investigation pointed to increased proportions ofpyrites; Ni^S^FeO^, alloy) and apatite particles alkane and decreased proportions of secondary(Ca5(F,Cl,OH)(PO4)3), and the occurrence of alcohol and diterpenic alcohol in the epicuticularph^'llosphere micro-organisms in the area (Turunen wax of declining Norway spruce trees {Picea abies L.et al, 1994; Back et al., 1996; Schreiber, 1996; Karst) (Prugel, Loosveldt & Garrec, 1994). Alkanes,Turunen & Huttunen, 1996) probably also however, are only minor constituents ( < 4 % ) ofcontributed to some of the variation observed in Scots pine epicuticular wax analysed here,needle wettability. The present investigation indicated that S de-

The responses of conifer needle surfaces to heavy position, especially H2SO4, most probably plays ametals are not well known, and research thus far has more important role in needle surface deteriorationdrawn attention to the mechanical abrasion of than Cu and Ni. It was also shown that pollutant-epicuticular wax structures and increased frequency induced changes in epicuticular wax structure andof occluded epistomatal chambers (Mankovskae^ a/., needle wettability mimic accelerated natural wax1988; Turunen et al., 1994). Insoluble heavy metal ageing, but the changes in wax chemical compositionparticles are not toxic, but if these initially insoluble might also be caused by pollutant-induced metabolicparticles are dissolved and dissociated as a result of changes in young, developing needles,increased acidity within the water film or droplets onthe needle surface, their effects on epicuticular wax

. . . 1 /T - 1 inrr-. ir s ACKNOWLEDGEMENTS

might be more pronounced (Little, 197J; Krause &Kaiser, 1977; Milne et al., 1990). In this regard, it is The authors wish to thank the staff of the Kevo Subarcticinteresting that the present long-term experiment on Research Station of the University of Turku, the De-subarctic Scots pines indicated that irrigation with partment of Biology, Botany and the Institute of ElectronCUSO4 and NiSO^ at pH 5-7 (alone), or in com- Optics of the University of Oulu, the Arctic Centre of thebination with H.SO^ at pH 3-1, did not mduce any Umversky of Lapland, the Finnish Forest Research

, , , . , . , , . , . . , Institute, Rovaniemi, and the Canadian forest Service,marked physicochemical changes m the epicuticular p^^^^^^^^^^ Canada. We also thank Mrs Virpi Alenius,wax, although there were signs of a reduction m ^ s Paivi Huhtala, Ms Elvi Hiltula, Mr Kyosti Kovaniemi,epistomatal WTD and the production of di-esters ^j^. p j j ^ Valikangas and Ms Tanya Zakrison for their

following metal treatment. The mechanisms by skilful assistance during the course of this work. Thewhich Cu " and Ni + penetrate the cuticle include authors also thank Mr Mervyn Lewis, Long Ashtondiffusion (concentration gradient) and ion exchange Research Station, Bristol, UK, for undertaking the GC-(electric gradient), in which valency plays an im- MS analyses. The investigation was financed by theportant role (Tyree, 1994). Following the dis- Finnish Ministry of Agriculture and Forestry, the Uni-sociation of metal sulphates into metal ions and versity of Lapland, the Jenny & Antti Wihuri Foundation

sulphates, the released divalent metal cations (Nl^^ ^"d the Academy of Fmland.

Cu "* ) probably penetrate the cuticle much more

slowly than monovalent protons (H" ) derived from j^gpgRENCESthe H2SO4 (Percy & Baker, 1989). The fact thatH,SO4 treatment did not increase needle wettability Back J, Turunen M Ferm A. Huttunen S 1996. Needle

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