ACTA BIOLOGICA SLOVENICA
LJUBLJANA 2009 Vol. 53, Št. 1: 35-45
ABS
Response of an ozone indicator plant before and after installation of a desulphurization device at a thermal power plant
Odziv bioindikatorskih rastlin za ozon pred in po namestitvi odžveplevalne naprave na termoenergetskem objektu
Nataša Kopušara*, Zdenka Mazeja, Franc Batičb aERICo Velenje; Koroška 58, 3320 Velenje, Slovenia Corresponding author. Tel: +386 3/ 898 1954; fax: +386 3/ 898 1942 E-mail address: natasa.kopusar@erico.si (N. Kopušar) ^University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
Abstract: The main goal of the research was to compare the plant response to air pollutants in the rural environment (Zavodnje village) before and after the installation of a desulphurisation device at unit 5 of the Šoštanj Thermal Power Plant (Slovenia). The installation of the cleaning device for SO2 in the year 2001 caused very significant reduction of annual average SO2 emission and immission, while concentrations of dust particles, O3, NOx, and CO2 in Zavodnje remained unchanged. On the other hand the average concentrations of ozone during seasonal experimental period increased after 2001, but there were no significant differences in AOT40. The impact of O3 in combination with other air pollutants was studied at Zavodnje in the period 1996-2003 using white clover (Trifolium repens 'Regal') on the basis of visible ozone injuries of leaves, biomass reduction and by analysis of chosen biochemical stress parameters in leaves - the content of plant pigments and antioxidants. The effects of reduction of SO2 after 2001 were observed in the biochemical responses of white clover plants, which showed better vitality. Despite of that, there was no significant difference in the level of plant injury by ozone between the period 1996-2000 and period 2001-2003. Biomass ratio CN-S/CN-R was also unaffected.
Key words: air pollution, desulphurization devices, Trifolium repens 'Regal'; ozone, sulphur dioxide, plant pigments and antioxidants, ozone injuries, biomass
Izvleček: Glavni namen raziskave je bil primerjava odziva rastlin na zračna onesnažila v ruralnem okolju (vas Zavodnje) pred in po namestitvi odžveplevalne naprave na 5. bloku Termoelektrarne Šoštanj. Namestitev te naprave v letu 2001 je povzročila značilno zmanjšanje letnih povprečnih imisij in emisij SO2, medtem ko se koncentracije prašnih delcev, O3, NOx, and CO2 v Zavodnjah niso spremenile. Na poskusnem mestu, blizu ANAS postaje v Zavodnjah, smo v obdobju 1996-2003 spremljali vpliv O3 v kombinaciji z drugimi zračnimi onesnažili na deteljo (Trifolium repens 'Regal') preko ocenjevanja vidnih ozonskih poškodb na listih detelje, sprememb v biomasi in analizah rastlinskih barvil in antioksidantov v listih. Učinek zmanjšanja koncentracije SO2 v okolju po letu 2001 se je na rastlinah pokazal na biokemijskem nivoju, medtem ko ni bilo učinka na zmanjšanje poškodb na listih ter biomasi.
Ključne besede: onesnaženost zraka, odžveplevalne naprave, Trifolium repens 'Regal'; ozon, žveplov dioksid, rastlinska barvila in antioksidanti, ozonske poškodbe, biomasa
Introduction
Increasing emission of air pollutants (NOx, VOCs) from traffic, thermal power plants (NOx, SO2) and transboundary transport are the main source of photo-oxidants. Sulphur dioxide (SO2) pollution in Slovenia was limited to larger towns and valleys around industrial and power plants (the Zasavje region, Mežica valley and Šalek basin). After the year 2001, when the new desulphurisa-tion device on the Šoštanj thermal power plant (ŠTPP) (Šalek basin) was installed, over 95% of SO2 was removed (Vrtačnik and Ribarič-Lasnik 2001). Other pollutants, especially nitrogen oxides (NOx) and hydrocarbons (VOCs), remained a problem and became increasingly problematic with increasing traffic. Nitrogen oxides and hydrocarbons participate in many photochemical reactions in the atmosphere, the results of which are photo-oxidant ozone (O3) and a number of organic compounds. O3 travels with air masses, rarely reacting at the place of its formation, but more often far away (Krupa and Manning 1988). The most exposed places are rural areas, where the main plant production takes place. Pollution of air with O3 occurs in summer, during the main growing period. As a strong oxidant, O3 is highly reactive with various forms of organic molecules in living systems and is extremely harmful to organisms.
Different air pollutants can act synergisti-cally or antagonistically in the environment. The adverse effects of O3 and SO2 on organisms are more pronounced if they act together than if they act separately (Mehlhorn et al. 1986, Lepper 1992). The direct damaging effect of ozone is due to the generation of free radicals (Penarrubia and Moreno 1999, Iriti and Faoro 2008). Plants respond with a defence mechanism against free radicals, based on the ability of specific enzymes and the activity of antioxidants to prevent, reduce or stop chain oxidations (Perl-Treves and Perl 2002, Jones 2006, Halliwell and Gutteridge 2007). Ozone is unlikely to reach the chloroplast, but it nevertheless causes pigment bleaching and lipid peroxidation (Harbinson and Hedley 1993). On the other hand, the phytotoxic effect of sulphur dioxide consists mainly of acidification of cell compartments and formation of free radicals during oxidation of sulphite to sulphate (Rennenberg
and Polle 1994). Additional effect is inhibition of Rubisco, the major enzyme of carbon dioxide fixation (Malhotra and Khan 1985, Okpodu et al. 1996, Lee et al. 1997).
To defend against both pollutants, O3 and SO2, a strong constitutive antioxidative defence is required. The influence of air pollutants on organisms depends on many factors, such as climate, nutrients, predisposition and age (Krupa and Manning 1988). The use of bioindicators is an effective method for determining the effects of air pollutants on organisms under given environmental conditions and for specifying limiting and critical values for pollutant concentrations.
Numerous reports have documented the effects of mixtures of O3 and SO2 on visible injury and plant growth and yield. These effects have shown responses ranging from antagonistic to synergistic (Guderian 1985). O3 impacts often manifest themselves as reduced plant growth and altered carbohydrate allocation patterns at the level of the whole plant (Pell et al. 1994, Grantz 2003). Reduced partitioning of photosynthates from leaves to sink tissues such as roots is commonly observed (Barnes et al. 1998). These impacts of O3 on allocation have profound effects on root function, plant water relations and integration of whole plant function (Grantz 2003). In contrast to ozone, SO2 can act principally as a nutrient, as sulphur-containing compound are required by the plants. Absorbed SO2 is used to synthesize glutathione, which is translocated in the phloem to the roots (Penarrubia and Moreno 1999). Flagler and Youngner (1982) found that SO2 affected tall fescue, but not nearly as severely as O3. Number of tillers was significantly reduced by an interaction of the two pollutants. Neither O 3 nor SO2 had an effect singly, but at the high O3 level, the addition of SO2 caused an 18% reduction in number of tillers.
The main goal of this research was to compare the response of two clones of white clover NC-S (less O3 tolerant) and NC-R (more O3 tolerant) to air pollutants, before and after the reduction of SO2 pollution from the ŠTPP in the rural environment. We expected that after reducing SO2 emission, the synergistic effect of SO2 and O3 would be diminished; reducing the harmful effect on plants.
Materials and methods
Study area
The experiment took place from 1996 to 2003 in Zavodnje near Šoštanj (734 m above sea level, 46o25'37.51'', 14o59'39.14") near an automatic measurement station of the ecological information centre of the Šoštanj thermal power plant (ŠTPP), where SO2, NOx, NO2 and O3 immission concentrations, air temperature, relative air humidity, wind speed and direction are measured every 30 minutes. Zavodnje was chosen as a test site, because it is the only site within the area of influence of the ŠTPP that is situated in a rural environment, where the concentrations of O3 and its precursors have been measured for many years.
AOT40 - ozone biological impact
AOT40, cumulative indices of ozone exposure in the period of three months (May to July) were calculated to evaluate the biological impact, which represent the sum of the differences between the hourly O3 concentrations (in ppb) and a threshold value of 40 ppb, for each hour in the interval 08:00-20:00h (Directive 2002/3/EC in Official Journal of European Communities, L. 67, March 9, 2002).
Plant material and method of exposure
Two clones of white clover (Trifolium repens 'Regal'), variety NC-S (O3 sensitive clone) and NC-R (O3 tolerant clone) were chosen from the group of plants known to be indicators of ozone impact (Heagle et al. 1995). The plants were exposed to outdoor environmental conditions in the study area in Zavodnje. Every plant, which showed advanced symptoms of disease (Polyth-rincium trifolii Kunze, UromycesflectensLagerh.) and virus infection, was excluded from experiment. For control of aphids, corn earworm and armyworms, Japanese beetles and mites, plants were treated by bifenthrin (Talstar) or natural insecticide pyrethrin. For slug controls slug pellets (metaldehyde) were used. For fungal pathogen no fungicides were used, because it is believed that some of them (benomyl and triadimefon) offer some protection against ozone toxicity. Data of the
specific exposure periods and harvests are shown in Tab. 1. One plant cutting of each white clover clone was planted in 12 L pot in greenhouse and after rooting period placed in a field for whole experimental season. New plants were used each year, provided by the ICP-Vegetation coordinator, Bangor, UK. Pots had self-irrigation system. There were 20 replications of each clone (NC-S, O3 sensitive clone; NC-R, O3 tolerant clone) every year. The experiment was carried out according to the protocol of the ICP Vegetation programme (Hayes et al. 2006).
Determination of ozone effects
The extent of visible ozone injury (white dots on upper surface of clover leaflets) was assessed weekly by calculating the percentage of injured leaves in each pot. All leaves, which were completely expanded, were used for assessing the injury class, based on a 0 to 6 scale (0: no injures, 1: very slight injury, occurrence of the first symptoms, 2: slight injury, 1-5% of the leaves with slight injury, 3: moderate injury, 5-25% of the leaves with injury, 4: heavy injury, 25-50% of the leaves injured, 5: very heavy injured, 50-90% of the leaves injured, 6: total injury, 90-100% of leaves are injured).
Clover biomass of each clone was determined at each harvest as dry weight of the above ground plant parts. Harvests were made every 28 days+/- 2 days (Tab. 1). The cutting height was 7 cm from the soil. The plant material was then dried to constant weight in an oven at 80 oC. The yield loss due to ozone impact was calculated by the ratio of biomass of the two clones, NC-S/NC-R. The yield loss was then calculated on the pot surface area to give the yield loss per hectare.
Plant material (leaves, which were completely expanded) for the analysis of plant pigments (chlorophylls a and b, zeaxanthine and lutein) and antioxidants (ascorbic acid) were taken before 9 a.m. (Tab. 1). It was frozen in liquid nitrogen in the field and stored in a cool box during transport to the ERICo laboratory. Only leaf blades were used in biochemical analyses. Ascorbic acid and plant pigments were analysed according to Pfeifhofer (1989) and Tausz (1996). The pigment and antioxidant analyses were made using high-pressure liquid chromatography (HPLC).
Table 1: Experimental season and dates of sampling of plants for biochemical analysis and measurement of biomass in different years.
Tabela 1: Poskusna obdobja in datumi vzorčenja rastlin za biokemijske analize in meritve biomase v različnih letih.
Experimental season	Dates of samplings and harvest
31.5.1996 - 4.9.1996	1 st harvest	11.7.1996
	2nd harvest	7.8.1996
	3rd harvest	4.9.1996
20.5.1997 - 8.8.1997	1 st harvest	12.6.1997
	2nd harvest	11.7.1997
	3rd harvest	8.8.1997
20.5.1999 - 14.8.1999	1 st harvest	20.6.1999
	2nd harvest	16.7.1999
	3rd harvest	14.8.1999
24.5.2000 - 17.8.2000	1 st harvest	20.6.2000
	2nd harvest	19.7.2000
	3rd harvest	17.8.2000
19.6.2001 - 10.9.2001	1 st harvest	18.7.2001
	2nd harvest	10.8.2001
	3rd harvest	10.9.2001
20.6.2002 - 18.9.2002	1 st harvest	23.7.2002
	2nd harvest	23.8.2002
	3rd harvest	18.9.2002
13.5.2003 - 8.8.2003	1st harvest	16.6.2003
2nd harvest	9.7.2003
3rd harvest	8.8.2003
0.5 g lyophilised and ground leaves was used for determining the amount of ascorbic acid. Extraction was carried out in 5 mL of 1.5% meta-phos-phoric acid (HPLC grade), and hexadecyltrimethy-lammonium bromide was used as the ion-pairing reagent. Ascorbic acid was detected directly at 248 nm. For determination of pigments little CaCO3 to neutralize the solution, and 5 ml of acetone (HPLC grade) were added to 1 g of ground white clover leaves. After centrifugation (10 min, 4000 circ/ min), supernatant was filtered through 0,45 ^m regenerated-cellulose-membrane filter into dark phial (Pfeifhofer 1989). Zeaxanthine and lutein were detected at 430 nm, chlorophyll a at 430 nm and chlorophyll b at 458 nm (Tausz 1996). For all analysis three replicates of samples were measured.
Statistical analysis
Statistic analysis made use of Statistic for Windows 5.5. Data analysis was based on parametric statistical methods (t-test, ANOVA/MANOVA and LSD test).
Results and Disscussion
The yearly emissions of air pollutants from the ŠTPP and average yearly immissions of air pollutants at Zavodnje are listed in Tab. 2, together with the production of energy in the power plant in the period before and after the installation of the desulphurization device at unit 5. After 2000, the amount of energy produced in the ŠTPP increased considerably while, at the same time, the amount of SO2 emissions per unit of energy produced was substantially reduced. Yearly concentrations of SO2 at Zavodnje as well as SO2 immission concentrations per amount of energy produced have decreased considerably since the desulphurization devices started operating. Annual concentrations of O3 and the amount of dust particulates at Zavodnje have not significantly changed in the periods under comparison. There was no change in the emissions of NOx and CO2. Average immission concentrations of pollutants at Zavodnje, given in Tab. 3, are calculated only for the period of experiments (experimental season)
each year. The average immission concentration of O3 at the experimental place in Zavodnje was between 1996 and 2000 73 ng/m3; concentrations then increased significantly to an average value of 91.7 ^g/m3 in the period of 2001- 2003. The results of A0T40 values, calculated for the period May-July, showed similar trend with higher values in the period after the installation of the desulphurization device at unit 5, but differences was not statistical significant. Latter can be explained by better climate conditions for the ozone formation, based on higher air temperatures, since there were no differences between concentrations of O3 precursors (NOx (NO+NO2) and NO2) in the periods before and after the desulphurisation device started operating (Tab. 3). Ambient ozone concentration in experimental site was above the levels defined as phytotoxic for vegetation by EU Directive 92/72/CEE. Critical levels of A0T40 for protection agricultural plants for three month (May - July) is 3 000 ppb (Sanders et al. 1995). Levels ofA0T40 (May - July) (Tab. 3) were above
critical values during all experiment years, with increasing potential, indicating a serious potential threat for crops at Zavodnje and surroundings.
The contents of chlorophylls in the leaves of both clones of white clover increased significantly after installation of the desulphurization device (Fig. 1a,b,c). The most obvious increase occurred in chlorophyll a (NC-R: from 1.86 mg/g DW before 2001 to 6.46 mg/g DW after 2001 and NC-S: from 1.77 mg/g DW before 2001 to 6.06 mg/g DW after 2001), what shows better vitality of plants after 2001. The processes involved in photosynthetic pigment alterations are partly triggered by exposure to elevated ozone but also to reduction of sulphur dioxide emission (Alonso et al. 2001). Beckerson and Hofstra (1979) found that in white bean the chlorophyll loss was greater in the pollutant combination (O3+SO2) than from the individual pollutants.
Before 2001 there were not differences in the content of ascorbic acid between NC-S and NC-R clones, but after the desulphurisation device
Table 2: Energy production and annual emission of air pollutants from ŠTPP and average concentration of air pollutants at the rural site Zavodnje before (1996-2000) and after (2001-2003) installation of a desulphurization device at unit 5 (Rotnik and Ribarič Lasnik 2004).
Tabela 2: Proizvodnja energije in letne emisije iz TEŠ ter povprečne koncentracije zračnih onesnažil v ruralnem območju Zavodnje pred (1996-2000) in po (2001-2003) namestitvi odžveplevalne naprave na blok 5 (Rotnik in Ribarič Lasnik 2004).
Annual average values		BEFORE (ä ± std)	AFTER (ä ± std)	(1)p
Energy produced in ŠTTP (MWh)		3884836±191219	4359106± 159231	*
Emission of air pollutants	SO2	0.01297 ± 0.00095	0.00414 ± 0.00101	***
from ŠTTP per energy	NOx	0.00273 ± 0.00019	0.00268 ± 0.00016	NS
produced (tons/MWh)	CO2	0.90981 ± 0.01581	0.93878 ± 0.05818	NS
	Dust particulate	0.00041 ± 0.00020	0.00012 ± 0.00002	NS
Average annual values of	SO2	0.00982 ± 0.00128	0.00414 ± 0.00066	***
air pollutants in Zavodnje	no2	0.00164 ± 0.00018	0.00122 ± 0.00023	NS
per energy produced (^g/ m3/GWh)	NOx	0.00200 ± 0.00029	0.00152 ± 0.00030	NS
	O3	0.01709 ± 0.00117	0.01679 ± 0.00189	NS
	Dust particulate	0.01462 ± 0.00917	0.01477 ± 0.00943	NS
	(mg/m2day/MWh)			
Average annual values of	SO2	38.2 ± 5.72	18.0 ± 2.65	**
air pollutants in Zavodnje	no2	6.40 ± 0.89	5.33 ± 1.15	NS
(^g/m3)	NOx	7.80 ± 1.30	6.67 ± 1.53	NS
	O3	66.4 ± 5.90	73.0 ± 6.24	NS
	Dust particulate	56.1 ± 33.5	65.3 ± 43.0	NS
	(mg/m2 day)			
(1) NS - not significant, * =	p<0.05, ** = p<0.01, ***	= p<0.001.		
Table 3: Average imission concentrations of air pollutants, air temperature and relative humidity during experimental season before (1996-2000) and after (2001-2003) installation of the desulphurization device at unit 5 (no data in 1998).
Tabela 3: Povprečne imisijske koncentracije zračnih onesnažil, temperature zraka in relativne vlage med trajanjem poskusa pred (1996-2000) in po (2001-2003) namestitvi odžveplevalne naprave na blok 5 (manjkajo podatki za leto 1998).
	BEFORE (ä ± std)	AFTER (ä ± std)	tdf, p(1)
Air temperature (oC)	16.5 ± 3.34	17.6 ± 4.44	t820=-3 92 ***
Relative humidity (%)	71.3 ± 10.42	67.8 ± 13.3	1820=4 14 ***
SO2 (^g/m3)(2)	23.2 ± 24.4	12.31 ± 13.0	1806=7 36 ***
NO2 (^g/m3)(2)	4.46 ± 3.34	4.89 ± 6.33	t807=-1.25
NOx (^g/m3)(2)	5.01 ± 3.95	5.77 ± 7.40	t807=-1.89
O3 (^g/m3)(2)	72.9 ± 26.9	91.7 ± 19.8	t815=-10 7***
A0T40 (ppb) (May-July)	16,355 ± 6,288	22,565 ± 5,803	t6 =-1.39
Legend: (1) df = degree of freedom; t = Student's t-test; * = p<0.05, ** = p<0.01, *** = p<0.001 (2)Source of half an hour's data: Ecological informative system of ŠTPP
Table 4: Ozone injuries on leaves of ozone-sensitive (NC-S) and ozone-resistant (NC-R) clones of the 'Regal' white clover (Trifolium repens 'Regal'), biomass and economic losses before (1996-2000) and after (2001-2003) installation of the desulphurization device at unit 5 (no data in 1998).
Tabela 4: Ozonske poškodbe na listih na ozon občutljivem (NC-S) in na ozon rezistentnem (NC-R) klonu plazeče detelje 'Regal' (Trifolium repens 'Regal'), biomasa in ekonomska ocena škode pred (1996-2000) in po (2001-2003) namestitvi odžveplevalne naprave na blok 5 (manjkajo podatki za leto 1998).
	Clone(5)	BEFORE (ä ± std)	AFTER (ä ± std)	tdf, Fdf, p(4)
Injury class (0-6)	NC-S NC-R	3.00 ± 0.56 0.58 ± 1.38	2.39 ± 1.65 0.19 ± 0.56	FU9=0.65 FU9=1.47
Frequency of ozone injuries in experimental period (%)(6)	NC-S NC-R	90.0 ± 11.5 16.6 ± 19.2	83.3 ± 28.8 11.1 ± 19.2	F15=0.18 F1>5=0.14
Biomass ratio	NC-S/NC-R	0.87 ± 0.13	0.90 ± 0.09	F9-21=0.52
Estimation of economic losses	(%)	13.5 ± 13.5	10.1 ± 8.7	t5=0.38
	(ton/ha) (23	0.33 ± 0.33	0.13 ± 0.11	t5=1.02
	(EUR/ha)(1)	21.4±20.9	8.35±7.24	t5=1.02
Legend: (1) Based on the price for 1 kg hay, which was 0.064 EUR (Statistical information, 281/04 (http://www.stat.si)
(2)	Surface of pots were 0.091 m2
(3)	Based on the average harvest for all experiment years - 2 t/ha
(4)	df = degree of freedom; t = Student's t-test; F = one-way ANOVA; * = p<0.05, ** = p<0.01, *** = p<0.001
(5)	NC-S clone - clone sensitive to ozone; NC-R clone resistant to ozone
(6)	Percentage of observations, when ozone injures on leaves were recorded through whole experimental period
a)
Chlorophyll a
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Sum of Chlorophylls
NC-S NC-R NC-S NC-R n=93 n=88 n=41 n=31
Before
After
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Chlorophyll a/b ratio
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NC-S NC-R n=93 n=87
Before
NC-S NC-R n=41 n=31
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F=12.4***, df1=3, df2=248
F=117.5***, df1=3, df2=249
F=83.2***, df1=3, df2=249
6
b
b
Figure 1a-d: Chlorophyll a (a), chlorophyll b (b), sum of chlorophylls (c) concentration (mg/g DW) and chlorophyll a and b ratio (d) in the leaves of (NC-S) and (NC-R) clones of the 'Regal' white clover (Trifolium repens 'Regal'), exposed in Zavodnje before and after installation of the desulphorization device at unit 5 at ŠTPP.
(Mean ± SD, F=One-way ANOVA, n= number of samples, df=degree of freedom, df1=df Effect, df2=df Error, ***, P<0.001, **, P<0.01, *, P<0.05) Different letters indicate significant differences (LSD) among clones.
Slika 1a-d: Koncentracije klorofila a (a), klorofila b (b), vsote klorofilov a in b (c) (mg/g ST) in razmerje klorofil a/klorofil b (d) v listih klonov NC-S in NC-R plazeče detelje 'Regal' (Trifolium repens 'Regal') v Zavodnjah, pred in po namestitvi odžveplevalne naprave na blok 5.
(Povp. ± SD, F=Enosmerna ANOVA, n= število vzorcev, df=stopnja prostosti, df=df učinka, df2=df napake, ***, P<0.001, **, P<0.01, *, P<0.05). Različne črke označujejo značilne razlike (LSD) med klonoma.
a)
2.5
£1.5
i= 1
О 0.5
Lutein
F=14.59***, df1=3, df2=249
NC-S NC-R n=93 n=88
Before
NC-S n=41
NC-R n=31
After
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Ascorbic acid
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NC-S n=80
NC-R n=71
Before
NC-S n=41
NC-R n=31
After
F=11.28***, df1=3, df2=219
b
a
a
b
Figure 2a-c: Lutein (a), zeaxanthin (b) and ascorbic acid (c) concentration (mg/g DW) in the leaves of ozone-sensitive (NC-S) and ozone-resistant (NC-R) clones of the 'Regal' white clover (Trifolium repens 'Regal'), exposed in Zavodnje before and after installation of the desulphorization device at unit 5 at ŠTPP (see key for details).
(Mean ± SD, F=One-way ANOVA, n= number of samples, df=degree of freedom, dfj=df Effect, df2=df Error, ***, P<0.001, **, P<0.01, *, P<0.05) Different letters indicate significant differences (LSD) among clones.
Slika 2a-c: Koncentracije luteina (a), zeaksantina (b) in askorbinske kisline (c) (mg/g DW) v listih klonov NC-S in NC-R plazeče detelje 'Regal' (Trifolium repens 'Regal') v Zavodnjah, pred in po namestitvi odžveplevalne naprave na blok 5.
(Povp. ± SD, F=Enosmerna ANOVA, n= število vzorcev, df=stopnja prostosti, dfj=df učinka, df2=df napake, ***, P<0.001, **, P<0.01, *, P<0.05). Različne črke označujejo značilne razlike (LSD) med klonoma.
on ŠTPP started operating, NC-S clone showed higher oxidative stress than NC-R clone (Fig. 2c). The content of ascorbic acid increases with intensity of oxidative stress (Foyer et al. 1997, Šircelj et al. 1997). Also the amount of lutein and zeaxantin (Fig. 2a,b) increased after 2001 at both clones. These results would indicate that the exposure to both O3 and SO2 before 2001 might diminish the protection ability of plants to respond to environmental stresses.
Despite of better vitality of plant and higher antioxidant defence ability after 2001, there were no significant differences in the levels of plant injury by ozone between the period 1996-2000 and period 2001-2003, only slight decrease in NC-R clone occurred (Tab. 4). Luwe et al. (1993) ascertained that antioxidants are not always accessible in times of stress. Examples of this are the limited availability of ascorbate in the apoplastic space during attack by the pollutant ozone (the very poor diffusion of ascorbate across the tylakoid membrane that provides ascorbate for the violax-anthin deepoxidase reaction) (Foyer et al. 1990), and the absence of effective antioxidants at the PSII reaction centre to prevent the oxidative damage associated with photo-inhibition (Arora et al. 2002).
The injury class of white clover leaves and frequencies of ozone injuries remained similar after 2001, in the case of NC-R clone a slight improvement can be seen, but not significantly proven (Tab. 4). It could be consequence of smaller synergistic effect of SO2 and other pollutants (NOx, NO2), lower relative humidity and higher temperatures after the year 2000 (Tab. 3). Higher average temperatures and lower relative humidity
both induce higher stomatal closure, which makes plants less sensitive to ozone (Benton et al. 2000). The same conclusion can explain why the reduction in SO2 immission concentration and simultaneous increase in O3 concentration were reflected in the absence of significant changes in NC-S/NC-R clones biomass ratio (Tab. 4). Moreover, the yield loss of NC-S clone was slightly lower after construction of the desulphurization device, but no statistically significant improvement was observed. The positive effect of lower SO2 emissions from the ŠTPP on yield losses (in proportion, yield per hectare and costs per hectare) of the white clover clones was also not significant.
Since most of the ozone absorbed through the stomata is decomposed rapidly, chlorosis does not appear to be a primary result of ozone exposure, but rather a secondary effect due to impaired photosynthetic capacity (Heath and Taylor 1997). Actually, there is strong evidence that other factors (phenological stage, nutritional state, co-occurrence with other pollutants and climatic elements) in addition to O3 pollution influence the biomass ratios (Fuhrer and Achermann 1999, Nali et al. 2009).
Acknowledgement
The authors are grateful to Prof. dr. Roger Pain for critical reading of the manuscript. This research was a part of the project financed by the Šoštanj Thermal Power Plant and by the Slovenian Research Agency (ARRS), Ljubljana.
References
Alonso, R., Elvira, S., Castillo, F. J., Gimeno, B.S., 2001. Interaction effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis. Plant, Cell Environ., 24, 905-916.
Arora, A., Sairam, R.K., Srivastava, G.C., 2002. Oxidative stress and antioxidative system in plants. Curr. Sci.82, 1227-1238.
Barnes, J. D., Davison, A. W., Booth, T.A., 1998. Ozone accelerates structural degradation of epicu-ticular wax on Norway spruce needles. New Phytol., 110, 309-318.
Beckerson, D. W, Hofstra, G., 1979. Stomatal responses of white bean to O3 and SO3 singly or in combination. Atmos. Environ., 13, 533-535.
Benton J., Fuhrer, J., Gimeno, B.S, Skirby, L, Palmer-Brown, D., Ball, G., Roadknight, C., Mills, G., 2000. An international cooperative programme indicates the widespread occurrence of ozone injury on crops. Agric. Ecosyst. Environ., 78, 19-30.
Directive 2002/3/EC of the European Parliament and of the Council of 12 February 2002 relating to ozone in ambient air. Official Journal of the European Communities, L 67, pp. 14-30.
Flagler, R. B., Youngner, V.B., 1982. Ozone and sulphur dioxide effects on tall fescue: I. Growth and Yield Responses. J. Environ. Qual., 11, 386-389.
Foyer, C. H., Furbank, R. T., Harbinson, J., Horton, P., 1990. The mechanisms contributing to photosyn-thetic control of electron transport by carbon assimilation in leaves. Photosynth. Res., 25, 83-100.
Foyer, C. H., Lopez-Delgado, h., Dat, J., Scott, I. M., 1997. Hydrogen peroxide and glutathioune associated mechanisms of acclamatory atress tolerance and signalling. Physiol.Plant., 100, 241-254.
Fuhrer, J., Achermann, B., 1999. Critical levels for ozone - level II. In: Fuhrer, J., Achermann, B. (eds.): Environmental Documentation, 115. Swiss Agency for the Environment, forests and Landscape, Bern. 333 pp.
Grantz, D.A., 2003. Ozone impacts on cotton: towards an integrated mechanism. Environ. Pollut., 126, 331-344.
Guderian, R., 1985. Air Pollution by Photochemical Oxidants. Formation, Transport, Control, and Effects on Plants. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo.
Halliwell, B., Gutteridge, J. M. C., 2007. Free Radicals in Biology and Medicine, 4th edition, Oxford University Press, New York.
Harbinson, J., Hedley, C. L., 1993. Changes in P-700 oxidation during the early stages of the induction of photosynthesis. Plant. Physiol.103, 649-660.
Hayes, F., Mills, G, Harmens, H., Novak, K., Williams, P., 2006. ICP Vegetation experimental protocol for monitoring the incidences of ozone injury on vegetation. Natural Environment Research Council, pp. 1-28. web site. http://icpvegetation.ceh.ac.uk/.
Heagle, A. S., Miller, J. E., Chevone, B. I., Dreschel, T. W., Manning, W. J., McCool, P. M., Lynn Morrison, C., Neely, G. E., Rebbeck, J., 1995. Response of a white clover indicator system to tropospheric ozone at eight locations in the United States. Water Air Soil Pollut., 85, 1373-1378.
Heath R. L., Taylir G. E. Jr, 1997. Physiological processes and plant responses to ozone exposure. In: Sandermann, H., Wellburn, A. R., Heath, R. L. (eds.), Forest Decline and Ozone. Springer-Verlag, Berlin, pp. 317-368.
Iriti, M., Faoro, F., 2008. Oxidative stress, the paradigm of ozone toxicity in plants and animals. Water Air Soil Pollut., 187, 285-301.
Jones, D.P., 2006. Redefining oxidative stress. Antioxid. Redox Signal.8, pp. 1865-1879.
Lee, E. H., Pausch, R. C., Rowland, R. A., Mulchi, C. L., Rudorff, B. F. T., 1997. Responses of field-grown soybean (cv. Essex) to elevated SO2 under two atmospheric CO2 concentrations. Environ. Exp. Bot., 37, 85-93.
Lepper, P., 1992. Wirkungen luftgetragener Schadstoffe (SO2, NO2, O3) auf antioxidative Systeme, Fettsauremuster und Frostresistenz von Kulturpflanzen. Wissenschafts-verlag Maraun, Frankfurt. 228 pp.
Luwe, M. W. F, Takkahama, U., Heber, U., 1993. Role of ascorbate in detoxifying ozone in the apoplast of spinach (Spinacia oleracea L.) leaves. Plant. Physiol., 101, 969-976.
Malhotra, S. S, Khan, A. A., 1985. Biochemical and physiological impact of major pollutants. In: Treshow, M. (Ed.), Air Pollution and Plant Life:, Wiley & Sons, New York, pp. 113-157.
Mehlhorn, H., Seufert, G., Schmidt, A., Kunert, K.J., 1986. Effect of SO2 and O3 on production of antioxidants in conifers. Plant Physiol., 82, 336-338.
Nali, C., Francini, A., Lorenzini, G., 2009. White clover clones as a cost-effective indicator of phyto-toxic ozone: 10 years of experience from central Italy. Environ. Pollut., 157, 1421-1426.
Okpodu, C. M., Alscher, R. G., Grabau, E. A., Cramer C. L., 1996. Physiological, biochemical and molecular effects of sulphur dioxide. J. Plant. Physiol.148, 309 - 316.
Pell, E. J., Temple, P. J., Friend, A. L., Mooney, H. A., Winner, W. E., 1994. Compensation as a plant response to ozone and associated stresses: an analysis of ROPIS experiments. J. Environ. Qual., 23, 429-436.
Penarrubia, L., Moreno, J., 1999. Molecular Mechanisms of Plant Responses to Elevated Levels of Tropospheric Ozone. In: Pessarakli, M. (Ed. ), Handbook of Plant and Crop Stress. Marcel Dekker, New York, Basel.
Perl-Treves R., Perl A. 2002. Oxidative stress: An introduction. In: Inzé D., Van Montagu M. (Eds.), Oxidative tress in Plants. Taylor & Francis, London and New York, pp. 1-32.
Pfeifhofer, H.W., 1989. On the pigment content of Norway spruce needles infected with Chrysomyxa rhododendri and the carotenoids of fungi aeciospores. Eur. J. Forest Pathol., 19, pp. 363-369.
Rennenber, H, Polle, A., 1994. Metabolic consequences of athmospheric sulphur influx into plant. In: Alscher, R. G, Wellburn, A. L. (Eds.), Plant Responses to the Gaseous Environment, Chapman & Hall, London, New York, pp. 165-180.
Sanders, G. E., Skärby, L., Ashmore, M. R., Fuhrer, J., 1995. Establishing critical levels for effects of air pollution on vegetation. Water Air Soil Pollut., 85, pp. 189-200.
Statistical information No. 281. Prices. No 46, 2004. Statistical Office of the Republic of Slovenia (20. Sept. 2004). Web site. http://www.stat.si (13. Oct. 2004)
Šircelj, H., Batič, F., Bienelli-Kalpič, A., 1997. Effects of ozone on pigment and ascorbic acid content in white clover (Trifolium repens L. cv. 'Menna') leaves. Acta biol. slov., 41, 4, 43-50.
Tausz, M., Kranner, I., Grill, D., 1996. Simultaneous determination of ascorbic acid and dehydroascorbic in plant materials by High-Performance Liquid Chromatography. Phytochem. Analysis, 7, 1-5.
Vrtačnik, J., Ribarič Lasnik, C., 2001. Ekološka sanacija TEŠ: 1987-2000. ERICo Velenje, 76 pp.