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The essential oil qualitative and quantitative composition in the needlesof Pinus sylvestris L. growing along industrial transects

Eugenija Kupcinskiene a,*, Aida Stikliene b, Asta Judzentiene c

a Department of Biology, Vytautas Magnus University, Vileikos 8, LT-44404 Kaunas, Lithuaniab Department of Ecology, Lithuanian University of Agriculture, Studentu 11, LT-53361 Kaunas, Akademija, Lithuania

c Institute of Chemistry, A. Gostauto 9, LT-01108 Vilnius, Lithuania

Received 1 February 2008; accepted 5 February 2008

Quantities of components of essential oil in the needles of Scots pine growing around twofactories are described in relation to pollution.

Abstract

The aim of this study was to evaluate composition of the essential oils in the needles of Pinus sylvestris growing in the areas affected by acement factory (CF), and an oil refinery (OR). Volatile components of the needles were analyzed by GC and GC/MS. The most heavily pollutedCF stand had significantly higher concentration of g-Terpinene, Caryophyllene oxide in the current-year needles, while higher concentration ofd-3-Carene, a-Terpinene, g-Terpinene and Terpinolene was documented for 1-year-old needles. The most heavily polluted OR stand hada significantly higher concentration of Sabinene þ b-Pinene, 1-epi-Cubenol in the current-year needles and a significantly higher concentrationof Camphene, Sabinene þ b-Pinene, Myrcene, a-Cadinene, 1-epi-Cubenol in the 1-year-old needles than the least polluted site. Along transectsan increase in the amount of some diterpenes and a decrease in the components of the shorter chain essential oils was observed. These effectscould be at least partially attributed to SO2.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Terpenes; Phytoindication; Conifers; Secondary metabolites; Air pollution; Cement dust pollution; Sulphur dioxide

1. Introduction

In many developed countries atmospheric pollution de-creased in the 1980s (Emberson, 2003; Wieser et al., 2006)and in the Central and Eastern European countries in the1990s (Mankovska, 1996; Staszewski et al., 1998; Sopaus-kiene et al., 2001). Despite this, during the past two decadesthere has been a growing interest in air pollutionevegetationeffects (Bytnerowicz et al., 2006). Up-to-date field studies,monitoring, and modelling work document air pollution andclimate change impacts on forests in Europe and NorthAmerica (Manninen and Huttunen, 1995; Krupa and Legge,2000; Bytnerowicz et al., 2002; Manning and Godzik, 2004;

Huttunen 2005; Huttunen and Manninen, 2005; Paoletti,2005, 2006; Gunthardt-Goerg and Vollenweider, 2006; Allenet al., 2007; Grulke et al. 2007). It is particularly challengingto identify specific or unique indicators for stresses.

Recently greater attention is paid to the secondary metabo-lites including essential oils of various plant species (Law-rence, 1991; Shu and Lawrence, 1997; Barnola and Cedeno,2000; Lawrence, 2001, 2006). Terpenes in conifers are signif-icant chemomarkers of environmental impact (Supuka andBerta, 1998). Diurnal (Barnola et al., 1997), seasonal andannual (Nault, 2003), climatic and edaphic (Kainulainenet al., 1992; Barnola et al., 1997), geographicalelatitudinalealtitudinal (Manninen et al., 1998, 2002; Nault, 2003) varia-tions of the concentration of terpenes in the needles of conifershave been documented.

Data concerning the effect of various anthropogenic factorson secondary compounds of the conifers is still controversial

* Corresponding author. Tel.: þ1 370 6122 3391.

E-mail address: e.kupcinskiene@gmail.com (E. Kupcinskiene).

0269-7491/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2008.02.001

Available online at www.sciencedirect.com

Environmental Pollution 155 (2008) 481e491www.elsevier.com/locate/envpol

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(Barnola and Cedeno, 2000; Zavala and Ravetta, 2002; Turtolaet al., 2006). Elevated CO2 concentration caused an increase inthe concentration of a-pinene (Heyworth et al., 1998; Sallaet al., 2001), while influence of elevated UV-B radiation onsecondary compounds of the conifers was not observed (Tur-tola et al., 2006). Increased limonene emission rates inozone-fumigated woody plants were described (Klusia et al.,2002). Wider surveys of the conifer essential oil compositionunder acidic and alkaline pollutant effects are still scarce(Heller et al., 1990; Fuksman, 2002; Kainulainen et al.,1992; Supuka and Berta, 1998). The potential for air pollutioneffects on biogenic VOC emissions should be considered whenassessing forest health (Erisman et al., 2005; Cape, 2007;Heath, 2007).

In Lithuania, since the 1990s the reduced level of emissionsin a cement factory (operating since 1952), and an oil refinery(since 1980) caused further adverse changes in the surround-ing forests (Kupcinskiene, 2001; Kupcinskiene and Huttunen,2005) and some disturbances were registered in the ongoingdecade (Ceburnis et al., 2002; Kupcinskiene, 2006; Judzen-tiene et al., 2007).

The present study was aimed to evaluate whether presentlevels of industrial pollution caused by the oil refinery andthe cement factory affects the qualitative and quantitativecharacteristics of the essential oils in the needles of Scotspine (Pinus sylvestris L.) growing under different levels ofpollution and at different distances from the pollution source.

2. Materials and methods

2.1. Study area

The Scots pine (Pinus sylvestris L.) growing along the transects from the

cement factory (CF) and the oil refinery (OR) in Lithuania were investigated.

In 2004e2005, total emissions from the CF comprised up to 3000 t year�1 and

emissions from the OR 24,000 t year�1 (data provided by the Ministry of En-

vironment of Lithuania). The total deposition of calcium and magnesium ions

along the CF transect ranged between 19.1 and 2.6 kg ha�1 month�1 (Armo-

laitis et al., 1999). Elevated concentrations of heavy metals in the mosses

were documented near CF and OR (Ceburnis et al., 2002). Sites near the

OR differed in the amounts of sulphur dioxide up to 6.7 times (with the highest

mean monthly concentration of 27 mg m�3; in 2005, being lower, 9.4 mg m�3),

and in the amounts of nitrogen dioxide up to 2 times (with the highest mean

monthly concentration of 6 mg m�3, registered in 2005). Sites near the CF dif-

fered in the amounts of sulphur dioxide up to 9 times (with the highest mean

monthly concentration of 8.3 mg m�3, registered in 2005; Kupcinskiene,

2006), and in the amounts of nitrogen dioxide up to 1.9 times (with the highest

mean monthly concentration of 3 mg m�3). In both transects, the most polluted

sites were the ones closest to the factories. The furthest stands were used as

control sites. Before sampling severe drought was registered (Table 1).

Middle-aged pines (51e62 years old) growing on histosols (near the CF),

and 46e75-year-old trees growing on luvisols (near the OR) were examined.

Selection of sites was based on availability of the stands and the prevailing

wind direction from each pollution source. Related to the CF, four stands be-

longing to Carico-sphagno-Pinetum (siccata) type in a 10-km transect (north-

east direction) were studied. Near the OR, four stands of Oxalido-Pinetum type

in a 5.4-km northeast transect and one stand (3.6 km away from the OR, east

direction) was examined.

2.2. Plant material

Branches facing the wind coming from the factory were cut from eight

pines at the height of 6e8 m above the ground (the third lowest part of the

crown). From each tree, four shoots with the current-year (c) and 1-year-old

(c þ 1) needles were used. July (10/07/2005) was chosen for sampling due

to the most vigorous growth of the new needles that expanded in June. For

analysis, four independent samples were prepared from each stand. A separate

sample was made by mixing equal amounts of the needles collected from eight

trees (Schulz et al., 1998) and drying the material at room temperature (20e

25 �C). Defoliation (expressed in %) of the sampled trees was estimated

according to EU standards (Anonymous, 1989).

2.3. Oil isolation and analysis

Oil yield was conducted by hydrodistillation of 50 g of dry needles. Pale

yellow oils were obtained in 0.2e0.6 and 0.2e0.3% (v/w) of the yield, respec-

tively, in the current-year and 1-year-old needles on a dry mass basis. GC anal-

ysis was done by an HP 5890(II) chromatograph equipped with FID and

capillary column HP-FFAP (30 m � 0.25 mm i.d., film thickness 0.25 mm).

Analyses by GC/MS were performed using an HP 5890 chromatograph inter-

faced to an HP 5971 mass spectrometer (ionization voltage 70 eV) and equip-

ped with a capillary column CP-Sil 8 CB (50 m � 0.32 mm i.d., film thickness

0.25 mm). Other details of oil analyses were described earlier (Mockute et al.,

2003; Mockute and Judzentiene, 2004). Qualitative analysis was based on

a comparison of retention times, indices with mass spectra libraries (Wiley

and NBS 54K), and other corresponding data (Adams, 2001). Quantity of

each component of essential oils was calculated as relative concentration

(peak area percentage) and amount (mg g�1 d.m., recalculated according to

the internal standart).

2.4. Statistical analysis

To compare the stands along the transects, dispersion and correlation anal-

yses were applied using EXCEL, SPSS, and SAS packages. Error bars in the

figures indicate an interval of 95% confidence. The normality of the data dis-

tribution was assessed and log transformations were performed. Data were

analysed with two-way analysis of variance. The significance of differences

between sites was assessed by Tukey’s multiple range test. Variances of con-

centration logarithms with their components (site, needle age and their inter-

action) were estimated by maximum likelihood (Schleppi et al., 2000).

3. Results

3.1. Needle essential oil general characteristics

There were no significant differences between the standsalong separate transects in the total yield of the essential oilsin the needles. Seventy identified components made up to89.1e95.1% of total oil content. The qualitative compositionof the main components appeared to be constant in the needlesof all pine stands investigated. However, there were consider-able differences in the amounts of separate components.The predominant fraction was found to be monoterpenes(19.0e40.0%), with the major constituents being a-pinene

Table 1

Precipitation (sum within decades, in mm) and temperature (mean values for

the decades) in JuneeJuly 2005, according to the data of the closest to the

transects Meteorological Station (Telsiai, Lithuania)

Month Decade Precipitation, mm Temperature�C

June I 3.1 11.9

II 0.6 15.8

III 2.0 16.0

July I 1.1 19.0

482 E. Kupcinskiene et al. / Environmental Pollution 155 (2008) 481e491

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(260e1256 mg g�1) and d-3-carene (190e980 mg g�1; Table 2).Among oxygenated monoterpenes (0.4e1.6%), bornyl acetatewas the most dominant constituent (10e237 mg g�1). Sesqui-terpenes formed 16.7e32.4% of the oils; amounts of mainsesquiterpenes (E )-caryophyllene and d-cadinene were 125e310 mg g�1 and 95e330 mg g�1, respectively. a-Cadinol(125e380 mg g�1) and epi-a-cadinol with epi-a and a-muuro-lols (120e453 mg g�1) were the major compounds in theoxygenated sesquiterpenes fraction (11.1e27.5%). Diterpenescomprised 0.9e13.5% of the oils. Phenols were the smallestgroup (0.2e2.0%).

3.2. The case of the cement factory

Comparison of the two most contrasting sites (stands grow-ing in 0.5 km and 10 km distance from the cement factory) ofthe transect in regard to pollution showed that the most heavilypolluted stand (at a distance of 0.5 km from the factory) hadsignificantly ( p < 0.05) lower concentration of bornyl acetate

(0.07 times of concentration in %), manoyl oxide (0.5 times),diterpene with RI 2105 (0.36 times) and higher concentrationof g-terpinene (4.3 times) and caryophyllene oxide (2.3 times)in current-year needles (Fig. 1), while significantly ( p < 0.05)higher concentration of d-3-carene (1.8 times), a-terpinene(2.6 times), g-terpinene (1.6 times) and terpinolene (2.0 times)was documented for 1-year-old needles (Fig. 2).

For needles of both ages, the concentration of somemonoterpenes was increasing and the concentration of oxy-genated monoterpenes, oxygenated sesquiterpenes and diter-penes was decreasing with the increasing defoliation of thetrees (Table 3). For current-year needles, the concentrationof some monoterpenes was decreasing and the concentrationof remainder terpenes was increasing with the increasing SO2.

For a-pinene, sabinene þ b-pinene, (E )-b-ocimene, n-un-decane, 2-decanone, n-decanol, 2-undecanone, (2E,4E )-deca-dienal, a-copaene, b-cubebene, b-copaene, aromadendrene,a-muurolene, d-cadinene, trans-cadina-1(2),4-diene,caryophyllene oxide, 1-epi-cubenol, unknown1 and

Table 2

Ranges of site mean values of the amount of the components (mg g�1 d.m.) of the essential oils in current-year and 1-year-old needles of Pinus sylvestris L. stands

growing at different distance from the factories (transects from the cement factory and the oil refinery, July 10, 2005)

Compound RIa Ranges of the mean

values for sites

Compound RIa Ranges of the mean

values for sites

Tricyclene þ a-Thujene 927 3e19 b-Copaene 1431 0.1e20

a-Pinene 939 260e1256 b-Gurjunene 1432 0.1e52

Camphene 953 62e186 Aromadendrene 1441 0.1e29

Sabinene þ b-Pinene 980 21e94 trans-Muurola-3,5-diene 1454 0.1e30

Myrcene 991 20e85 a-Humulene 1455 30e89

d-3-Carene 1012 190e980 cis-Muurola-4(14),5-diene 1467 0.1e11

a-Terpinene 1018 12e112 g-Muurolene 1480 26e191

p-Cymene 1026 0.1e18 Germacrene D 1485 44e140

Limonene þ b-Phellandrene 1031 16e95 b-Selinene 1490 10e46

(Z )-b-Ocimene 1037 0.1e19 trans-Muurola-4(14),5-diene 1495 0.1e4

(E )-b-Ocimene 1050 8e105 Bicyclogermacrene 1500 45e185

g-Terpinene 1060 7e50 a-Muurolene 1500 19e110

Terpinolene 1089 41e166 trans-b-Guaiene 1513 0.1e60

n-Undecane 1100 0.1e6 g-Cadinene 1514 51e164

Borneol 1169 0.1e20 d-Cadinene 1523 95e330

p-Mentha-1,5-dien-8-ol 1170 0.1e5 trans-Cadina-1(2),4-diene 1535 4e75

Terpinen-4-ol 1177 12e46 a-Cadinene 1539 6e60

m-Cymen-8-ol 1180 0.1e5 a-Calacorene 1546 0.1e15

p-Cymen-8-ol 1183 0.1e10 b-Calacorene 1566 0e6

a-Terpineol 1189 3e38 (Z)-3-Hexenyl benzoate 1567 0.1e48

2-Decanone 1192 0.1e5 Germacrene D-4-ol 1576 19e160

n-Dodecane 1200 0.1e9 Spathulenol 1578 6e66

Thymol, methyl ether 1235 0.1e9 Caryophyllene oxide 1580 0.1e50

n-Decanol 1270 0.1e10 Gleenol 1587 0.1e21

Bornyl acetate 1289 10e237 b-Oplopenone 1608 0.1e25

2-Undecanone 1294 0.1e14 1,10-di-epi-Cubenol 1619 2e39

(2E,4E )-Decadienal 1300 0.1e19 1-epi-Cubenol 1629 11e45

d-Elemene 1338 20e97 epi-a-Cadinolþ 1640

Terpinyl acetate 1349 19e63 epi-a-Muurolol þ a-Muurolol 1642 120e453

a-Cubebene 1351 0.1e12 a-Cadinol 1654 125e380

a-Copaene 1377 0.1e38 Eudesma-4(15),7-die-1b-ol 1688 0.1e8

b-Bourbonene 1386 0.1e25 Manoyl oxide 1988 3e42

b-Cubebene 1388 0.1e4 Abietadiene 2088 0.1e40

b-Elemene 1391 37e100 Unknown1 2105 15e350

Tetradecane 1400 0.1e16 Abieta-8(14),13(15)-diene 2154 0.1e18

(E )-Caryophyllene 1419 125e310

a RI, retention index.

483E. Kupcinskiene et al. / Environmental Pollution 155 (2008) 481e491

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abieta-8(14),13(15)-diene, the main source of variation (Table4) was site and for the biggest part of the other compoundsneedle age differences were greater compared to sitedifferences.

3.3. The case of the oil refinery

Comparison of the two most contrasting sites of the transectin regards to pollution showed that the most heavily pollutedstand (at a distance of 2.0 km from the factory) had a signifi-cantly ( p < 0.05) higher concentration of sabinene þ

b-pinene (1.8 times), 1-epi-cubenol (2.2 times) and lower con-centration of manoyl oxide (0.1 times), diterpene with RI 2105(0.4 times) in the current-year-needles (Fig. 3) than the leastpolluted site (located at 5.4 km away from the oil refinery).Also, the 1-year-old needles of the most polluted site pre-sented a significantly ( p < 0.05) higher concentration of cam-phene (3.1 times), sabinene þ b-pinene (3.2 times), myrcene(2.4 times), a-cadinene (2.5 times), 1-epi-cubenol (2.4 times)and a lower percentage of manoyl oxide (0.1 times) and diter-pene with RI 2105 (0.2 times) than those sampled at the leastpolluted site (Fig. 4).

γ-Terpinene

020406080

0.5 1.5 3.5 10Distance, km

μg g

-1

μg g

-1

μg g

-1

μg g

-1

μg g

-1

Caryophyllene oxide

01020304050

0.5 1.5 3.5 10Distance, km

Manoyl oxide

0.1

10.1

20.1

30.1

0.5 1.5 3.5 10Distance, km

Unknown (RI 2105)

0100200300400

0.5 1.5 3.5 10Distance, km

Bornyl acetate

-50

0

50

100

150

0.5 1.5 3.5 10Distance, km

Fig. 1. Concentration (mg g�1 d.m.; mean values, n ¼ 4) of the components of essential oils in current-year needles of Scots pine (Pinus sylvestris L.) stands

growing at different distance from the cement factory (July 10, 2005). Error bars indicate an interval of 95% confidence.

δ-3-Carene

0

500

1000

1500

0.5 1.5 3.5 10Distance, km

μg g

-1

α-Terpinene

020406080

100

0.5 1.5 3.5 10Distance, km

γ-Terpinene

05

10152025

0.5 1.5 3.5 10Distance, km

μg g

-1

Terpinolene

0

50

100

150

200

0.5 1.5 3.5 10Distance, km

μg g

-1

μg g

-1

Fig. 2. Concentration (mg g�1 d.m.; mean values, n ¼ 4) of the components of essential oils in 1-year-old needles of Scots pine (Pinus sylvestris L.) stands growing

at different distance from the cement factory (July 10, 2005). Error bars indicate an interval of 95% confidence.

484 E. Kupcinskiene et al. / Environmental Pollution 155 (2008) 481e491

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In current-year needles and 1-year-old needles, the con-centration of the most monoterpenes, some sesquiterpenesand oxygenated sesquiterpenes was increasing with the in-creasing defoliation of the trees/increasing concentration ofSO2 (Table 5), while opposite relations were found forditerpenes.

Along the oil refinery transect, the most heavily pollutedstand (a distance of 2.0 km from the factory) had the lowest(by a large degree) concentration of diterpenes in both cur-rent-year and 1-year-old needles.

For tricyclene þ a-thujene, a-pinene, camphene, myrcene,d-3-carene, (Z )-b-ocimene, (E )-b-ocimene, g-terpinene, n-un-decane, borneol, p-mentha-1,5-dien-8-ol, m-cymen-8-ol,p-cymen-8-ol, n-dodecane, thymol methyl ether, n-decanol,bornyl acetate, terpinyl acetate, n-tetradecane, cis-muurola-4(14),5-diene, g-muurolene, germacrene D-4-ol, trans-muur-ola-4(14),5-diene, a-muurolene, a-cadinene, caryophyllene

Table 3

Correlation coefficients between the concentration of the components of the

essential oils in current-year (c) and 1-year-old needles (c þ 1) and defoliation

of the Scots pine (Pinus sylvestris L.) stand/sulphur dioxide concentration

(transect from the cement factory; July 10, 2005)

Compound Defoliation SO2

c needles c þ 1 needles c needles c þ 1 needles

Monoterpenes

Myrcene n.s. n.s. �0.664** n.s.

a-Terpinene n.s. 0.722** �0.891** n.s.

Limonene þb-Phellandrene

n.s. n.s. �0.511* n.s.

g-Terpinene 0.827** n.s. n.s. 0.610*

Oxygenated monoterpenesBornyl acetate �0.814** n.s. n.s. n.s.

Borneol �0.507* n.s. n.s. n.s.

Terpinen-4-ol

Sesquiterpenes

a-Cubebene n.s. n.s. n.s. 0.734**

b-Cubebene n.s. n.s. n.s. 0.641**

a-Copaene �0.507* �0.617* n.s. �0.702**

a-Humulene 0.498* n.s. n.s. n.s.

b-Selinene 0.504* �0.652** 0.647** �0.621**

Bicyclogermacrene n.s. n.s. 0.684** n.s.

g-Cadinene 0.499* n.s. n.s. n.s.

a-Calacorene n.s. n.s. n.s. �0.513*

Oxygenated sesquiterpenes

Germacrene D-4-ol n.s. n.s. 0.619* n.s.

Spathulenol n.s. n.s. 0.616* n.s.

Caryophyllene oxide �0.721** n.s. 0.499* n.s.

1,10-di-epi-Cubenol n.s. n.s. 0.537* n.s.

1-epi-Cubenol n.s. n.s. 0.570* n.s.

epi-a-Muurolol þa-Muurolol

n.s. �0.540* n.s. n.s.

a-Cadinol �0.558* 0.565*

DiterpenesManoyl oxide �0.752** n.s. n.s. n.s.

Unknown1 �0.851** n.s. n.s. n.s.

n.s., non-significant ( p>0.05) correlations; *p < 0.05; **p < 0.01.

Table 4

Variances of concentration (of the components of essential oils in the needles

of Pinus sylvestris L. growing at different distance from the cement factory)

logarithms with their components (site, needle age, site � needle age interac-

tion) estimated by maximum likelihood (July 10, 2005)

Components of essential oils Site Age Site � Age Residuals

Tricyclene þ a-Thujene 0.131 1.560 0.148 0.554

a-Pinene 0.478 0.002 0.113 0.080

Camphene 0.034 1.034 0.008 0.124

Sabinene þ b-Pinene 0.145 0.450 0.076 0.087

Myrcene 0.221 2.525 0.403 0.269

d-3-Carene 1.673 2.813 0.635 0.102

a-Terpinene 1.717 1.767 3.623 0.271

p-Cymene 0.524 3.811 1.566 0.757

Limonene þ b-Phellandrene 0.180 0.335 0.292 0.103

(Z )-b-Ocimene 0.986 2.781 0.443 0.955

(E )-b-Ocimene 4.175 2.486 0.480 0.118

g-Terpinene 2.037 0.020 0.857 0.119

Terpinolene 1.363 0.947 0.779 0.084

n-Undecane 0.307 0.005 0.307 0.206

Borneol 1.742 3.509 2.096 1.002

p-Mentha-1,5-dien-8-ol 0.827 0.199 0.920 1.127

Terpinen-4-ol 0.119 0.365 0.903 0.227

m-Cymen-8-ol 0 0 0 0

p-Cymen-8-ol 0.147 1.207 0.722 0.965

a-Terpineol 0.113 5.163 0.011 0.207

2-Decanone 2.547 0.045 0.045 0.286

n-Dodecane 0.272 0.659 0.851 0.282

Thymol, methyl ether 0.710 0.410 0.463 1.186

n-Decanol 1.043 0.080 0.045 0.255

Bornyl acetate 2.184 4.921 0.363 0.190

2-Undecanone 2.163 0.325 1.377 0.443

(2E,4E )-Decadienal 2.481 0.465 0.321 0.902

b-Elemene 0.077 1.822 0.127 0.421

Terpinyl acetate 0.162 1.390 0.384 0.242

a-Cubebene 1.284 3.602 1.385 0.203

a-Copaene 2.831 0.472 0.477 0.388

b-Bourbonene 1.752 0.060 2.053 0.745

b-Cubebene 2.490 0.166 0.690 0.458

b-Elemene 0.079 0.000 0.254 0.143

Tetradecane 0.007 0.007 0.305 0.271

(E )-Caryophyllene 0.038 1.529 0.225 0.098

b-Copaene 1.184 0.551 0.072 0.900

b-Gurjunene 0.558 2.064 0.248 1.028

Aromadendrene 1.013 0.017 0.502 0.601

trans-Muurola-3,5-diene 0.408 0.663 0.573 1.202

a-Humulene 0.084 2.187 0.097 0.109

cis-Muurola-4(14),5-diene 0.039 0.318 0.064 0.612

g-Muurolene 0.441 3.268 1.080 0.432

Germacrene D 0.151 0.802 0.490 0.324

b-Selinene 0.066 0.254 0.736 0.088

trans-Muurola-4(14),5-diene 0.297 0.081 0.081 1.337

Bicyclogermacrene 0.142 1.979 0.427 0.123

a-Muurolene 0.548 0.281 0.444 0.150

trans-b-Guaiene 0.166 0.166 0.166 0.166

g-Cadinene 0.249 0.510 0.200 0.087

d-Cadinene 0.179 0.040 0.245 0.113

trans-Cadina-1(2),4-diene 0.838 0.043 0.394 0.796

a-Cadinene 0.183 1.353 0.201 0.181

a-Calacorene 0.603 0.425 2.594 0.665

b-Calacorene 0.324 1.913 0.678 1.169

(Z)-3-Hexenyl benzoate 0.335 3.205 1.527 0.671

Germacrene D-4-ol 0.259 9.869 0.273 0.109

Spathulenol 1.5663 17.106 0.587 0.261

Caryophyllene oxide 1.594 0.067 0.185 0.120

Gleenol 0.726 2.481 1.202 0.830

(continued on next page)

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oxide, 1-epi-cubenol, a-cadinol and unknown1, the mainsource of variation (Table 6) was the site.

4. Discussion

Varying amounts of terpene species in the needles of pineswere described by different research teams: 10 (Berta et al.,1997), 42 (Bojovic et al., 2005), 55 (Idzojtic et al., 2005),56 (De Simon et al., 2001); in our study 70 componentswere identified. Generally, our results concerning the compo-sition of the essential oils in the needles of P. sylvestris weresimilar to those reviewed by Lawrence (1991). Sixteen mono-terpenes are described among 10 pine species (Da Silva et al.,2001). In our Scots pine study 13 monoterpenes were identi-fied. Domination of monoterpenes and sesquiterpenes ob-served in our study are in agreement with other studies ofconifers (Dormont et al., 1998; De Simon et al., 2001; Manni-nen et al., 2002; Bojovic et al., 2005).

Decrease in the concentration of d-3-carene, terpinolene(near the cement factory), and an increase in the concentrationof camphene, sabinene þ b-pinene (near the oil refinery) wasdocumented in the needles of the pines growing closest to thefactories. Conifer changes in the needle a-pinene at thepolluted urban site (Berta et al., 1997; Supuka et al., 1997)

or under elevated CO2 concentration were reported (Heyworthet al., 1998; Salla et al., 2001).

Scots pine stands growing along transects from the factoriesin Lithuania differed significantly not only according to theconcentrations of some major (mentioned above) compounds,but also according to intermediate (camphene, myrcene, terpi-nolene, sabinene þ b-pinene, a-terpinene, bornyl acetate,d-elemene, b-elemene, g-muurolene, bicyclogermacrene, g-cadinene) and minor (g-terpinene, a-terpineol, a-cadinene,caryophyllene oxide, 1-epi-cubenol, manoyl oxide) compo-nents of essential oils in the needles. Continuous stress onthe pines due to the factories might induce, in part, the ob-served needle differences in quantities of separate componentsof the essential oils between stands growing along selectedtransects in Lithuania. This lends support to reported changesin the terpene production of several conifers under the effect ofcontaminated urban atmosphere (Juttner, 1988; Supuka andBerta, 1998).

Concentration correlations between air pollutants and theseparate components of essential oils in the needles corre-sponded to the data summed for monoterpenes, oxygenatedmonoterpenes, sesquiterpenes, oxygenated sesquiterpenesand diterpenes (Judzentiene et al., 2007). Along the CF tran-sect for current-year needles, the percentage of diterpeneswas decreasing with the increasing concentration of SO2

(r ¼ �0.573; p < 0.05). Along the OR transect, in both thecurrent-year and 1-year-old needles, the percentage of diter-penes was decreasing with the increasing SO2 (respectively,r ¼ �0.773, p < 0.01; r ¼ �0.486, p < 0.05); an opposite re-lation was true for sesquiterpenes (respectively, r ¼ �0.751,p < 0.01; r ¼ 0.785, p < 0.01). Similar to what was shownin many other countries, examinations of the industrial tran-sects in Lithuania showed that worsening of general tree con-dition could be related to the effects of the factories(Armolaitis et al., 1999; Kupcinskiene, 2001; Kupcinskieneand Huttunen, 2005). Defoliation in conifers has been associ-ated with changes in leaf primary and secondary metabolite

Table 4 (continued)

Components of essential oils Site Age Site � Age Residuals

b-Oplopenone 0.335 0.478 0.809 0.604

1,10-di-epi-Cubenol 0.161 0.115 0.543 0.384

1-epi-Cubenol 0.448 0.077 0.150 0.158

epi-a-Cadinolþepi-a-Muurolol þ a-Muurolol 0.606 0.950 0.358 0.166

a-Cadinol 0.341 1.119 0.209 0.114

Eudesma-4(15),7-1b-ol 0.3456 0.785 0.790 1.058

Manoyl oxide 0.486 2.620 1.075 0.098

Abietadiene 0.259 0.003 0.957 0.400

Unknown1 0.617 0.117 0.590 0.104

Abieta-8(14),13(15)-diene 2.110 0.019 1.733 0.731

Sabinene +β-Pinene

0

20

40

60

2.0 2.5 3.6 5.3 5.4

Distance, km

μg g

-1

μg g

-1

μg g

-1

μg g

-1

1-epi-Cubenol

-20

0

20

40

60

2.0 2.5 3.6 5.3 5.4

Distance, km

Manoyl oxide

0

10

20

30

40

2.0 2.5 3.6 5.3 5.4Distance, km

Unknown (RI 2105)

0100200300400500

2.0 2.5 3.6 5.3 5.4Distance, km

Fig. 3. Concentration (mg g�1 d.m.; mean values, n ¼ 4) of the components of essential oils in current-year needles of Scots pine (Pinus sylvestris L.) stands grow-

ing at different distance from the oil refinery (July 10, 2005). Error bars indicate an interval of 95% confidence.

486 E. Kupcinskiene et al. / Environmental Pollution 155 (2008) 481e491

Author's personal copy

chemistry (Wagner and Evans, 1985; Schonwitz et al., 1991);also relations between defoliation of conifers affected by in-sects and the concentration of terpenes in the needles weredocumented (Baranchikov et al., 1991), while some other ob-servations did not show a correlation between the percentageof needle loss and percentile terpene amounts in the needles(Schonwitz et al., 1991). Plant secondary metabolism relationswith defoliation caused by air pollution are poorly described.In our study, we observed a decrease in the concentration ofditerpenes in the needles for the stands (the nearest to the ce-ment factory and the oil refinery) that had the higher defolia-tion and shorter needle retention (Kupcinskiene, 2001;Kupcinskiene and Huttunen, 2005; Stikliene et al., 2006; Jud-zentiene et al., 2007). Among the stands of the selected indus-trial transects the extent of the differences for concentration ofseparate compounds of essential oils in the needles varied upto 10 times (transect from the cement factory) and also upto 10 times (transect from the oil refinery), while tree defolia-tion of the transects differed respectively 1.7, 1.8 and 1.3 timesand aerial pollutant concentrations differed along transects re-spectively 5.5 (SO2) and 1.8 (SO2) times. Variability of the

results (differences concerning distance, defoliation and SO2

effects) might be caused by several reasons. Due to differentresidence times for pollutants emitted from the factory ineach site, a special set (in terms of concentration) of toxiccompounds is formed. In addition to the SO2 effect, defolia-tion is a consequence of a variety of other stressors of anthro-pogenic and natural origin. Selection of sites was limited byavailability of pine stands, which is why only the furthest sitesof the transects corresponded exactly to the direction of theprevailing wind. Strong chemical reactivity of essential oilsalso might be an important source of variation.

Along two industrial transects the increase in the amount ofsome diterpenes and decrease in the components of the shorterchain essential oils was observed. These effects could be atleast partly attributed to sulphur dioxide that is omitted byboth factories. Changes in the concentration of some compo-nents of the essential oils in the needles of Scots pine werehigher to some extent compared to the differences in visible(defoliation of the trees) damage to the trees, and might bea useful tool for the detection of latent changes caused byindustrial, especially sulphur dioxide, pollution.

α-Cadinene

0

10

20

30

2.0 2.5 3.6 5.3 5.4Distance, km

Camphene

050

100150200250

2.0 2.5 3.6 5.3 5.4Distance, km

μg g

-1

μg g

-1

μg g

-1

μg g

-1

μg g

-1

μg g

-1

μg g

-1

Sabinene + β-Pinene

0

50

100

150

2.0 2.5 3.6 5.3 5.4Distance, km

Myrcene

020406080

100

2.0 2.5 3.6 5.3 5.4Distance, km

1-epi-Cubenol

0

50

100

150

2.0 2.5 3.6 5.3 5.4Distance, km

Manoyl oxide

0

20

40

60

80

2.0 2.5 3.6 5.3 5.4Distance, km

Unknown (RI 2105)

0

200

400

600

800

2.0 2.5 3.6 5.3 5.4Distance, km

Fig. 4. Concentration (mg g�1 d.m.; mean values, n ¼ 4) of the components of essential oils in 1-year-old needles of Scots pine (Pinus sylvestris L.) stands growing

at different distance from the oil refinery (July 10, 2005). Error bars indicate an interval of 95% confidence.

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In the Picea abies study diminished content of the needlemonoterpenes was registered under 14 months of treatmentby ozone and acid mist (Heller et al., 1990), although diter-penes were not estimated. In another study involving ozoneand acid mist, different terpene patterns existed but couldnot be connected with any of the experimental conditions(Schonwitz and Merk, 1986). Further evidence, especially in

controlled-condition experiments, is needed to test ourfindings.

Relations between nutrient availability and the secondaryplant compounds in Scots pine have been described in otherstudies (Barnola and Cedeno, 2000; Lavola et al., 2003).Our results obtained in Scots pine nutritional studies alongtransects from the cement factory and the oil refinery (Kup-cinskiene, 2006) are consistent with those that have reportedchanges in volatile terpenoid concentrations in the leaves ofseveral plant species growing in conditions with low nitro-gen and phosphorus availability (Barnola and Cedeno,2000).

Qualitative and quantitative terpene variations between co-nifer needles of different age have been described (Cariseyand Bauce, 1997; Helanova et al., 2006; Lagalante et al.,2006; Campbell and Taylor, 2007). Seasonal and annual(Nault, 2003) geographicalelatitudinalealtitudinal (Manninenet al., 1998, 2002; Nault, 2003) variations in the concentrationof terpenes in the needles of conifers might be attributed atleast partially to the needle age-related effects, although infor-mation concerning pollutant effects on terpenes in needles ofdifferent age is scarce. It is documented that altered nitrogensupply affects allocation to secondary metabolites differently,depending on the age of the needles (Kainulainen et al.,1996). According to the present data needle age was themain source of variation for the biggest part of the compo-nents of essential oils. In our study comparison of the needlesof two age classes revealed diverse reactions concerning thecomponents of essential oils affected by pollution accordingto the presence/absence of the reaction, direction (increase/decrease) of the effect, and the extent (changes in concentra-tion). Correlations between concentration of the componentsof the essential oils and tree defoliation depended on theage of the needles. Cluster analysis of the concentration ofessential oils in the needles applied for industrial transectsshowed larger effects of pollution for current-year needlescompared to 1-year-old needles in the case of pine standsnear the oil refinery (Judzentiene et al., 2007). Despite presentlow-level dust emissions from the cement factory, both needleage classes showed significant factory distance-relatedchanges, confirming the opinion about the absence of thresh-old for dust effects (Farmer, 1993; Stikliene et al., 2006). For-mer studies of needle surface erosion along the sameindustrial pine stand transects revealed a higher indicativevalue of the older-age needles (Kupcinskiene and Huttunen,2005), while some nutritional transformations were observedfor current-year needles (Kupcinskiene, 2006). The presentstudy provides evidence that in a polluted environment impor-tant biochemical changes occur within the first month afterneedle expansion. It is well known that younger needles aremore resistant than older ones to various stressors. Biggerchanges in essential oils for freshly expanded (less than1 month old) current-year needles might reflect their protec-tive reaction to acute effects of pollutants including SO2.Older needles might be more adapted for chronic exposureto pollutants and their defence aids might also be alreadyexhausted.

Table 5

Correlation coefficients between the concentration of the components of essen-

tial oils in current-year (c) and 1-year-old needles (c þ 1) and defoliation of

the Scots pine (Pinus sylvestris L.) stand/sulphur dioxide concentration (tran-

sect from the oil refinery, July 10, 2005)

Compound Defoliation SO2

c needles c þ 1 needles c needles c þ 1 needles

Monoterpenes

a-Pinene n.s. n.s. 0.447* n.s.

Camphene n.s. 0.737** n.s. n.s.

Sabinene þ b-Pinene 0.633** 0.730** 0.596** n.s.

Myrcene 0.555* 0.577** 0.445* n.s.

d-3-Carene n.s. �0.540* n.s. �0.598**

(Z )-b-Ocimene 0.478* n.s. n.s. n.s.

Limonene þ b-

Phellandrene

n.s. 0.520* 0.448* n.s.

(E )-b-Ocimene 0.591** 0.488* 0.585** n.s.

c-Terpinene n.s. 0.541* n.s. 0.466*

Terpinolene �0.571** 0.483* �0.589** n.s.

Oxygenated monoterpenes

Borneol 0.448* 0.444* n.s. n.s.

p-Mentha-1,5-dien-8-ol �0.513* n.s. n.s. n.s.

Terpinen-4-ol 0.608** n.s. 0.556* n.s.

m-Cymen-8-ol 0.594** 0.471* n.s. n.s.

p-Cymen-8-ol n.s. n.s. n.s. 0.596*

a-Terpineol 0.443* n.s. 0.499* n.s.

Bornyl acetate n.s. n.s. n.s. �0.554*

Terpinyl acetate 0.497* 0.751** n.s. 0.581**

Sesquiterpenes

b-Bourbonene 0.449 * n.s. n.s. n.s.

a-Cubebene n.s. �0.471* n.s. �0.471*

a-Copaene n.s. 0.522* n.s. 0.562*

b-Cubebene �0.471* �0.594** �0.471** �0.594**

b-Elemene n.s. n.s. 0.585** 0.682**

(E )-Caryophyllene n.s. n.s. n.s. 0.463*

Aromadendrene n.s. 0.654** n.s. 0.783**

a-Humulene n.s. n.s. n.s. 0.780**

g-Muurolene n.s. n.s. n.s. 0.674**

Germacrene D n.s. n.s. n.s. 0.490*

Bicyclogermacrene 0.627** 0.449* 0.751** 0.793**

a-Muurolene 0.683** 0.605** 0.610** 0.675**

c-Cadinene 0.629** n.s. 0.513* 0.460*

d-Cadinene 0.529* 0.636** 0.461* 0.746**

a-Cadinene n.s. 0.822* n.s. 0.797**

Oxygenated sesquiterpenesGermacrene D-4-ol 0.492* n.s. n.s. n.s.

Spathulenol 0.619** n.s. n.s. n.s.

1-epi-Cubenol n.s. 0.735** 0.446* n.s.

Diterpenes

Unknown1 �0.553* �0.914** �0.737** �0.521*

n.s., non-significant correlations; *p < 0.05; **p < 0.01.

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Because pollen and seeds of the pine are dispersed by wind,the role of the essential oils in the needles cannot be related tothe attraction of insects and might be quite unambiguouslyconnected with protection against pathogens and also para-sites. The essential oils or their components have been shownto exhibit antiviral, antibacterial, antimycotic, antitoxigenic,antiparasitic, and insecticidal properties, and activity againstmite pests and nematodes (Supuka and Berta, 1998; Barnolaand Cedeno, 2000). Monoterpenes in particular might be a fac-tor determining host plant resistance (Barnola et al., 1997;Tiberi et al., 1999; Chen et al., 2002; Petrakis et al., 2005).In our study, changes in concentration of some major, interme-diate or minor components of the essential oils of the needles,mentioned above, may modify tree relations with the sur-rounding biota including susceptibility of the polluted (by in-dustrial emissions) pine stands to the pathogens and parasites.

Higher production of the shorter-chain terpenes (observedalong the cement factory and oil refinery transects) might bea consequence of tree growth in the polluted environment re-quiring energy consumption for the reparation processes,while at the same time investing smaller resources for synthe-sis of protective compounds. A two-fold increase in monoter-pene and sesquiterpene accumulation in the needles has beenreported under methyl jasmonate treatment (Martin et al.,2003). This suggests the way in which phytohormones mightbe involved in the plant biochemical response to a pollutedenvironment.

Monoterpenes, coming from the conifer forests, form a sig-nificant part in BVOC emissions (Geron et al., 2000; Cape,2007). Alterations in concentration of major components ofthe essential oils like d-3-carene and terpinolene a-pinene inthe needles of the pines growing closest to the factories maycontribute to the changes in emissions of terpenes from thepolluted forests.

5. Conclusions

Under the effect of present-level industrial emissions,changes occur in the concentration of components of the es-sential oils in the needles of Scots pine. Significantly affectedby industrial emissions, the major components of essential oils

Table 6

Variances of concentration (of the components of essential oils in the needles

of Pinus sylvestris L. growing at different distance from the oil refinery) log-

arithms with their components (site, needle age, site � needle age interaction)

estimated by maximum likelihood (July 10, 2005)

Components of essential oils Source of variance

Site Age Site � Age Residuals

Tricyclene þ a-Thujene 1.115 0.368 0.550 0.587

a-Pinene 0.362 0.042 0.152 0.036

Camphene 0.380 0.006 1.072 0.095

Sabinene þ b-Pinene 1.058 1.928 0.390 0.115

Myrcene 0.956 0.452 0.199 0.100

d-3-Carene 0.364 0.114 0.081 0.027

a-Terpinene 0.199 0.196 0.276 0.434

p-Cymene 0.180 0.321 0.247 0.880

Limonene þ b-Phellandrene 0.281 0 0.326 0.123

(Z )-b-Ocimene 1.945 0.213 0.814 0.507

(E )-b-Ocimene 0.926 0.150 0.415 0.099

g-Terpinene 0.364 0.163 0.314 0.194

Terpinolene 0.453 0.989 0.515 0.145

n-Undecane 0.189 0.126 0.102 0.239

Borneol 2.376 0.417 0.417 0.806

p-Mentha-1,5-dien-8-ol 2.096 2.617 1.239 0.477

Terpinen-4-ol 0.218 6.072 0.483 0.193

m-Cymen-8-ol 3.314 0.133 0.133 0.309

p-Cymen-8-ol 0.919 0.007 1.542 0.973

a-Terpineol 1.197 4.560 0.817 0.467

2-Decanone 1.414 6.725 1.699 0.438

n-Dodecane 0.309 0.062 0.035 0.387

Thymol, methyl ether 2.660 0.023 0.048 0.892

n-Decanol 1.629 0.101 0.513 0.484

Bornyl acetate 1.230 0.527 0.495 0.132

2-Undecanone 0.444 0.632 0.334 0.360

(2E,4E )-Decadienal 0.004 0.236 0.223 0.261

d-Elemene 0.399 4.357 0.248 0.078

Terpinyl acetate 0.807 0.001 0.143 0.073

a-Cubebene 0.530 0.133 0.795 0.309

a-Copaene 0.721 0.578 2.382 0.269

b-Bourbonene 0.921 0.373 1.648 0.390

b-Cubebene 2.927 0.224 0.224 0.268

b-Elemene 0.672 1.412 0.102 0.044

Tetradecane 1.144 0.009 0.049 0.210

(E )-Caryophyllene 0.286 1.565 0.114 0.024

b-Copaene 0 0 0 0

b-Gurjunene 0.016 0.089 0.411 0.364

Aromadendrene 0.822 1.831 0.545 0.328

trans-Muurola-3,5-diene 0.244 2.121 1.814 0.719

a-Humulene 0.293 0.929 0.143 0.039

cis-Muurola-4(14),5-diene 1.502 0.843 0.881 0.794

g-Muurolene 1.447 0.006 0.383 0.083

Germacrene D 0.314 0.017 0.201 0.103

b-Selinene 0.099 1.370 1.352 0.149

trans-Muurola-4(14),5-diene 1.148 0.159 0.468 0.555

Bicyclogermacrene 0.487 3.417 0.101 0.029

a-Muurolene 1.386 0.011 0.200 0.090

trans-b-Guaiene 0 0 0 0

g-Cadinene 0.557 2.696 0.286 0.230

d-Cadinene 0.750 6.367 0.313 0.526

trans-Cadina-1(2),4-diene 0.170 0.769 0.462 0.586

a-Cadinene 0.527 0.124 0.212 0.177

a-Calacorene 0.890 1.634 0.382 0.962

b-Calacorene 0.343 0.065 0.396 0.595

(Z)-3-Hexenyl benzoate 0.489 7.893 0.255 0.671

Germacrene D-4-ol 0.218 15.334 0.131 0.153

Spathulenol 1.776 7.869 0.821 0.594

Caryophyllene oxide 1.041 0.097 1.121 0.542

Table 6 (continued )

Components of essential oils Source of variance

Site Age Site � Age Residuals

Gleenol 0.065 3.084 0.298 0.844

b-Oplopenone 0.459 0.932 1.654 0.511

1,10-di-epi-Cubenol 0.332 0.755 0.340 0.326

1-epi-Cubenol 0.518 0.455 0.238 0.120

epi-a-Cadinolþepi-a-Muurolol þ

a-Muurolol

0.468 0.757 0.044 0.015

a-Cadinol 0.415 0.095 0.067 0.071

Eudesma-4(15).7-die-1b-ol 0 0 0 0

Manoyl oxide 1.956 2.196 0.362 0.319

Abietadiene 0.491 0.224 0.985 0.453

Unknown1 3.414 0.490 0.382 0.056

Abieta-8(14),13(15)-diene 1.092 1.193 0.736 0.512

489E. Kupcinskiene et al. / Environmental Pollution 155 (2008) 481e491

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in the needles were d-3-carene, terpinolene (decrease near thecement factory), camphene, sabinene þ b-pinene (increasenear the oil refinery), and diterpenes (decrease near the cementfactory and the oil refinery). Correlation analyses showed re-lations between the concentrations of air pollutants and the to-tal concentration of the main classes of the essential oils ofseparate components of these classes. Current-year needleswere more informative indicators of the effects of pollutionthan 1-year-old needles in the case of the OR transect, andneedles of both ages were equally valuable in the case ofthe CF transect. In general, the pollution from the oil refineryor the cement factory had caused higher proportions ofshorter-chain terpenes and lower proportions of longer-chainterpenes. Along selected industrial transects the concentrationof separate compounds of essential oils in the needles of pinesvaried up to 10 times, while transect tree defoliation differedto lower extent (up to 1.8 times).

Acknowledgements

This work was supported by the Study and Science Founda-tion of Lithuania, Project ‘‘FIBISTRESS’’, grant No. C-05033/05. The authors thank Rima Potockiene for assistance in statis-tical analyses and Dr Almantas Kliucius for visual assessmentof tree conditions.

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