- Email: [email protected]
Effect of air traffic pollution on seed quality characteristics of Pinus brutia
Environmental and Experimental Botany 74 (2011) 157–161
Contents lists available at ScienceDirect
Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Effect of air traffic pollution on seed quality characteristics of Pinus brutia Petros Ganatsas a , Marianthi Tsakaldimi a,∗ , Georgios Zachariadis b a b
Laboratory of Silviculture, School of Forestry and Natural Environment, Aristotle University, Thessaloniki 54124, Greece Laboratory of Analytical Chemistry, School of Chemistry, Aristotle University, Thessaloniki 54124, Greece
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 17 May 2011 Accepted 19 May 2011 Keywords: Pine seeds Germination Seed viability Heavy metals Road traffic
a b s t r a c t This study was undertaken to examine to what degree traffic air pollution affect the reproductive material (the seeds) of Pinus brutia. The study focused on the investigation of the seeds morphological characteristics and physiological attitudes as well as heavy metal concentrations of P. brutia trees grown close the ring-road of Thessaloniki (Greece’s second biggest city), polluted by heavy traffic conditions, and how these characteristics are changed with the distance from the pollution source. A sampling procedure was established for seed collection from various distances from the pollution source. Seeds were properly prepared, measured for their morphological characteristics and heavy metal concentrations, as well as for their physiological behaviour (germination and viability). Analysis of the results showed that even though there were no significant differences in seed heavy metal concentrations, and only few in seed morphological characteristics, related to the distance from the traffic pollution source, there was a significant alteration of physiological behaviour for the seeds originated closed to the traffic. All the seed collected close to the ring-road (sample distances 0 and 30 m from the road) were unable to germinate, even those subjected to pre-treatments and repeated germination trials over longer duration, while those collected from distances over 100 m from the ring-road showed a common germination behaviour. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Air pollution affects human health as well as plants and microorganisms. The sampling use of plant tissues has long been shown to be an effective indicator of atmospheric pollution (Sawidis et al., 1995; Dursun et al., 2002). Vehicular traffic emissions play an important role in air pollution. They consist of gaseous pollutants like nitric oxides, carbon monoxide, sulphur dioxide, hydrocarbons, fine and coarse particulate matter like diesel soot and airborne particulate-bound trace metals and metals (De Fré et al., 1994; Laschober et al., 2004). Metallic particles released by motor vehicles may remain in the air for some time, but most of them get deposited on the roadside soils and the plant material close to the road. Metals can enter plants directly via rain and dust but also be taken up from the soil through roots (Jozic et al., 2009). Many trace metals, such as Pb, Zn, Ni, Cd, etc., are released into the atmosphere adjacent to roadways, affecting the roadside habitat (Wong et al., 1984). In recent years, it has been shown that heavy metal levels in soil and vegetation have increased considerably due to traffic pollution and the problem rises as daily traffic increases (Onder and Dursun, 2006). For example, Celik et al. (2005) reported concentration values of Fe, Pb, Cu, Mn in plants at roadsides approximately four times
∗ Corresponding author. E-mail address: [email protected] (M. Tsakaldimi). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.014
greater than values in plants at control sites. A review research published on metal pollution in Greek cities (included Thessaloniki city) reported increased concentrations of the metals Pb, Cu, Zn, Pt and Pd in urban environment such as top-soils of highways and streets. Also, the soil and plant analyses showed that Fe and Cu are the most accumulated elements in plants, while pollution from copper (Cu), iron (Fe) and cadmium (Cd) seems to be the most serious problem (Farmaki and Thomaidis, 2008). However, while some heavy metals perform important biological functions, such as being structural, and catalytic constituents of enzymes and other proteins (Hall, 2002), when in excess heavy metals are toxic to plant cells and may inhibit growth (Steffens, 1990; Hall, 2002). Many of accumulating species (those with resistance), are species with large crowns and broad leaves, although, many conifers have also been found to accumulate heavy metals (Bako et al., 2005). The reproductive organs of coniferous trees, with their complex organization and long duration of the reproductive cycle, are the most sensitive to the damaging influence of a wide range of anthropogenic contaminants (Geras’kin et al., 2005). Especially for pine species with a long maturation period (usually 24–36 months), may provide the opportunities for pollutants genotoxic effects and significant DNA damages in the undifferentiated stem cells, for individuals exposed in situ in polluted areas. Additionally, the presence of mutagenic factors near highways is undoubted; many components of exhaust gases, pavements, and technologic dust may be
158
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
mutagenic (Stvolinskaya, 2000), while mutagenesis is probably not the sole factor responsible for pathological seed development. The seeds as the reproductive organs of seed-bearing plants are especially sensitive to traffic pollutants; thus, in plants growing near highways with heavy traffic, seed viability decreases, and the number of abnormally developing embryos increases more than twice (Stvolinskaya, 2000). Analysing plant tissues can give better results in terms of sensitivity and reproducibility (Lau and Luk, 2001). Uptake of elements into plants can happen via roots from the soil and transported to the leaves and directly via the leaves from the air, or by precipitation. There are many investigations on heavy metal and other pollutant levels in air, and their toxic effect on the plants. Less well known, however, are the effects of chronic exposures on the reproductive capacity of forest trees, particularly in situations where visible injury does not occur. Even though, the effects of air pollutants on foliage of coniferous species are well known (Sawidis et al., 1995; Fuentes et al., 2007; Onder and Dursun, 2006), unfortunately, there are scarce studies dealt with the effect of air pollution on seed ecology of conifers in non-controlled environments. Fedorkov (1999) reported that the viability of Siberian spruce seeds was significantly reduced under severe deposition of sulphur dioxide and heavy metals (nickel and copper) in air. Also, Huston and Dochinger (1977) reported that previous studies on conifer species located in a polluted region or at various distances from cities, noted a marked reduction in cone dimensions, 1000-seed weight and germination capacity. Thessaloniki (Greece’s second biggest city) is considered one of the most polluted cities in Europe, especially with respect to airborne particles (Vlachokostas et al., 2010). The ring road of Thessaloniki separates the city from the sub-urban forest and bears high traffic volume (over 100,000 vehicles daily) with three lanes of traffic per direction. The roadsides characterized by high concentration of air pollutants CO, SO2 , NO2 , PM10 and O3 (Region of Central Macedonia, 2003) and high concentration of heavy metals in roadside dusts (max values of Pb, Cu and Mn 180, 300 and 280 mg/kg, respectively) (Ewen et al., 2009). This study was undertaken to examine to what degree air traffic pollution affect the reproductive organs (the seeds) of Pinus brutia in a non-controlled environment. The study focused on: (a) the investigation of the morphological and physiological characteristics and heavy metal content of seeds of P. brutia trees, grown near to the ring road of Thessaloniki city, and (b) taking into account that the concentration of heavy metals in plants decreases with the distance from the pollution source (Chonopoulos et al., 1997; Celik et al., 2005), how these characteristics are changed with the distance from the ring road.
2. Materials and methods 2.1. Experimental site The ring-road of Thessaloniki city is bordered by the sub-urban forest of the city named “Kedrinos Lofos”, separates the city from the sub-urban forest, and bears large traffic volume. The dominant tree species in the forest is the Mediterranean Pine, P. brutia, which in the great part of the area forms pure, even-aged stands coming from reforestation. The vegetation of the general area belongs to the Quercetalia pubescentis zone and especially to the Ostryo-Carpinion alliance. The soils of the area are slightly acid up to neutral, shallow up to middle depth, poor of nutritious ingredients, with a high percentage of stones and pebbles. The climate is a Mediterranean type with a cool winter and high temperature during the summer. According to meteorological station of Aristotle University of Thessaloniki, which is located close to the study sites, the mean
annually precipitation is 420 mm and the mean annual temperature is 15.6 ◦ C. The dry period lasts from the middle of May to the end of September. 2.2. Cone sampling and preparation of samples Three (3) uniform sampling sites (concerning topography and canopy cover) were chosen for the study, along the ring-road (Suppl. Fig. 1). For each sampling site, cone samples were collected in late Spring 2009 and at five distances from the road edge to the inner part of the forest (0, 30, 100, 500 and 1500 m). At each distance point, five (5) cones were collected from each of 10 selected trees (Reyes and Mercedes Casal, 2002) of P. brutia 50-year-old with similar dimensions (totally 750 cones). None of these trees displayed foliar pollution injury symptoms. All cones were already mature (i.e., brown-coloured). All the collected samples were put in polyethylene bags and transferred to the laboratory. Cone opening was achieved by thermal treatment of cones at 45 ◦ C in an oven, for three days. All seeds were extracted manually. For each sampling site the seeds were pooled for each distance point. Damaged and insect-infected seeds were discarded, and the empty ones were eliminated using the floating method in distilled water (Boydak et al., 2003; Ganatsas et al., 2008). The full filled seeds were stored in a refrigerator at 4 ◦ C. 2.3. Measurements of seed morphological characteristics In order to measure the seed size (length and width), weight and moisture content, 20 full filled seeds from each distance × 3 sampling sites were selected. These measurements were accomplished twice with the same number of sampled seeds. Totally 600 seeds were measured. Their dimensions were measured with callipers in accuracy 0.01 mm. Seeds were weighed with electrical balance of four decimal counters and then they oven-dried at 107 ◦ C for 17 ± 1 h and they weighted again (Paitaridou et al., 2006). The moisture content of seeds was calculated by measuring the weight lost after the seeds oven-drying (ISTA, 1996). The length of embryo (mm) was recorded on a random sample of 30 seeds (10 seeds × 3 sampling sites) from each distance, using a stereoscope (Leica). Photographs were taken on millimetre paper background by a digital photo camera (Nicon BM-7). 2.4. Chemical analysis Chemical analysis of seeds was carried out in order to determine the heavy metals contents in seeds. A sample of pooled seeds of each distance point per sampling site was oven dried at 80 ◦ C for 1 week and was ground to a fine powder by a microhammer mill in order to pass through a 1 mm aperture screen. From each powder sample, three subsamples of 1 g were weighted and, metals were extracted from each subsample by wet digestion method by the use of the acid mixture H2 SO4 + HNO3 + HClO4 . 1 g DW of each subsample was placed in an open quartz tube. Six and half (6.5) ml of the acid mixture (1 ml H2 SO4 + 5 ml HNO3 + 0.5 ml HClO4 ) were added to each tube and the mixture was left 2 h at room temperature. It was warmed for 2 h at 50 ◦ C and then heated at 180 ◦ C for 4 h (Sawidis et al., 1995). Then the solutions were filtered through Whatman type 589/2 filters and the filtrate was diluted to 50 ml volume with extra pure water. Each final solution was put in cleaned plastic containers and analyzed for lead (Pb), chromium (Cr), copper (Cu), iron (Fe) and manganese (Mn) concentrations by Atomic Absorption Spectrometry (Perkin Elmer, 5100 ZL) and Inductively Coupled Plasma Atomic Emission Spectrometry (Perkin Elmer, Optima 3100 XL Plasma Spectrometer). The heavy metals contents are given as g/g dry weight (DW).
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
159
Table 1 Effects of distance, from the ring road edge (pollutant source) to the inner part of the forest, on seed morphological characteristics of P. brutia. Distance from the ring road (m)
Seed length (mm)
Seed width (mm)
Seed fresh weight (g)
Seed dry weight (g)
Seed moisture content (%)
Length of embryo (mm)
0 30 100 500 1500
6.92 (0.26) b 6.88 (0.25) b 6.54 (0.19) b 6.94 (0.15) b 7.66 (0.14) a
3.95 (0.13) b 4.03 (0.09) b 3.96 (0.09) b 4.26 (0.07) ab 4.37 (0.08) a
0.057 (3 × 10−3 ) b 0.055 (3 × 10−3 ) b 0.061 (1 × 10−3 ) b 0.057 (2 × 10−3 ) b 0.070 (3 × 10−3 ) a
0.053 (3 × 10−3 ) b 0.050 (3 × 10−3 ) b 0.057 (1 × 10−3 ) b 0.053 (2 × 10−3 ) b 0.065 (3 × 10−3 ) a
7.20 (0.93) ns 7.36 (0.99) ns 7.63 (0.53) ns 7.49 (0.37) ns 6.23 (0.49) ns
5.8 (0.4) ns 5.8 (0.3) ns 6.0 (0.4) ns 6.2 (0.4) ns 6.5 (0.5) ns
Values of the tested variables are the averages ± the standard error of the mean (in brackets). Within a column, the averages followed by different letter are significantly different, P ≤ 0.05. ns: non significant differences.
2.5. Seed germination and viability experiments 2.5.1. 1st experiment Germination tests were performed in 9-cm-diameter glass petri dishes on two layers of filter paper saturated with distilled water (Ganatsas et al., 2008). Three replications of 25 seeds were used for each distance point and for each sampling site; thus, a total of 225 seeds were used for each distance point (3 replications of 25 seeds × 3 sampling sites). The seeds were immersed in 3% (v/v) formaldehyde/deionized water for 5 min to avoid fungal contamination. After that, the seeds were washed with deionized water. Filter papers were changed every 3 d in order to reduce the chance of cross-contamination by micro-flora (Ganatsas and Tsakaldimi, 2007). Experiments were carried out in a temperature controlled growth chamber at 20 ± 0.5 ◦ C with a photoperiod of 12 h and luminance of 1000 lux (Boydak et al., 2003). Seed germination was measured every 2nd day for 28 d duration, and germination was considered to have occurred if the radical protruded 5 mm from the seed coat. Seeds with abnormal radicals were recorded but they were excluded from the germination counts. Cumulative germination percentage was evaluated every 2 days and the final value was obtained after 28 d. Mean germination time (MGT) and the peak value (PV) were also calculated as these parameters combined with the germination capacity are show better the seed performance. MGT was calculated according to the following A D +A D2 +...AnDn formula:MGT = 1 1A +A2 +...An where A is the number of seeds 1 2 germinating per day, D is the time corresponding to A in days and n is the number of days to final count (Ganatsas et al., 2008; Korkmaz and Korkmaz, 2009). Peak value (PV) is the maximum mean daily germination obtained by dividing the maximum cumulative percentage reached at any time during the test period by the number of days from sowing when that maximum was reached (Swaminathan et al., 1993). 2.5.2. 2nd experiment In the second experiment, new samples of seeds from the sampling distances, that showed null germination, were subjected to cold–moist stratification for two months at a temperature of 2–4 ◦ C in the dark and after that to mechanical scarification with sandpaper (Skordilis and Thanos, 1995). The germination tests were performed as in the previous experiment. At the end of the test, the seeds that were not germinated were subjected to a Tetrazolium test (1% 2,3,5-triphenyl tetrazolium chloride) to determine their viability (ISTA, 1996). If these ungerminated seeds were viable then other reasons would be responsible (e.g. experimental conditions) for germination failure. In the case of the Tetrazolium test, only those seeds that show a significant respiratory activity (dark-red), after 24 h staining period, are considered as viable. 2.6. Statistical analysis √ Percentages data were transformed using the arcsine transformation to meet the assumptions of normality and homoscedasticity. Tables and figures present untransformed data and standard
error of the mean (±SE). One-way ANOVA was used to test differences. Duncan’s test was used to compare the mean values of the studied seed variables among the 5 sampling distances. All statistical analyses were conducted using a critical P value ≤ 0.05. 3. Results 3.1. Seed morphological characteristics Seed overall morphological characteristics are shown in Table 1. Seed dimensions of trees from the inner part of the forest (at 1500 m from the ring road edge), were significantly greater (7.66 mm length and 4.37 mm width) than that of the trees were closer to the ring-road edge. Seed dimensions of trees were 0, 30, 100 and 500 m away from the ring-road, did not show any significant differences. The mean seed fresh and dry weight of the studied seeds was fluctuated from 0.057 to 0.070 g and from 0.050 to 0.065 g respectively, while the significantly greatest values were observed in seeds of trees abstained 1500 m from the ring-road. However, between the studied seeds, non-significant differences were observed in seed moisture content that ranged from 6.23 to 7.63%. The length of seed embryo was ranged from 5.5 to 6.5 mm and was slightly greater in the seeds at the distances 500 and 1500 m, but no significant differences were detected. 3.2. Seed heavy metal content The obtained results from the heavy metal analysis in the studied seeds of P. brutia are given in Table 2. The heavy metals content in seeds, collected at various distances from the ring-road edge to the inner part of the sub-urban forest, generally did not show significant differences, with few exceptions. Fe content was found significantly greatest in seeds of trees near to the ring-road edge (0 m away), while Mn content was found significantly lowest in seeds collected from the trees existed in the longest distance from the road edge (1500 m away). However, Pb content of the seeds was found always lower than the detection limit of 1 g/g DW. 3.3. Seed germination behaviour and viability According to the results from the first experiment, there was a significant reduction in seed germination of P. brutia as the distance from the ring-road decreased (Fig. 1). The highest seed germination capacity (84%) was observed in trees located 1500 m away from the ring-road (towards the heart of the sub-urban forest). There were no significant differences on seed germination capacity between the trees located at 100 and 500 m away from the ring road; their seed germination was fluctuated between 61 and 63%. Between the above three distances there was no variation in time taken for the initiation of seed germination (germination started 12 days after sowing) and no significant differences were observed for the PV and MGT (Fig. 1, Table 3). However, at distances 0 and 30 m, the seed germination was completely inhibited and no seeds germinated.
160
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
Table 2 Content of Pb, Cr, Cu, Fe and Mn (g/g DW) in seeds of P. brutia. Distance from ring road edge (m)
Pb
Cr
Cu
Fe
Mn
0 30 100 500 1500
<1 <1 <1 <1 <1
1.467 (0.89) ns 0.923 (0.13) ns 1.006 (0.13) ns 0.984 (0.14) ns 1.230 (0.22) ns
11.21 (1.04) ns 9.60 (0.58) ns 8.50 (1.09) ns 8.36 (0.43) ns 10.12 (0.44) ns
135.05 (15.9) a 108.86 (2.2) b 94.38 (4.2) b 95.17 (5.2) b 108.95 (12.3) b
33.72 (2.80) a 36.83 (1.63) a 37.78 (1.21) a 34.93 (2.29) a 22.68 (1.43) b
Values are the averages (g/g DW) from 9 replications ± the standard error of the mean (in brackets). Within a column, the averages followed by different letter are significantly different, P ≤ 0.05. ns: non significant differences. Table 3 Effect of distance, from the ring road (pollutant source) to the inner part of the forest, on mean germination time (MGT, days) and peak value (PV) of P. brutia seeds. Distance from the ring road (m)
MGT PV
0
30
100
500
1500
0.0 0.0
0.0 0.0
23.8 (2.8) ns 2.18 (0.43) ns
24.4 (2.9) ns 2.24 (0.41) ns
24.0 (2.5) ns 3.00 (0.59) ns
Values are the mean and the standard error of the mean (in brackets). ns: non significant differences, P > 0.05.
The results from the second experiment, studying the seeds from the sampling distances 0 and 30 m from the ring-road, which subjected previously to cold–moist stratification and to mechanical scarification, confirmed that the germination capacity of these seeds was null. Similarly, the tetrazolium test revealed that these seeds were not viable at all, after 24 h staining period. 4. Discussion
Germination %
According to the results analysis, the distance from the traffic pollution source (ring-road of Thessaloniki city) significantly affected some morphological characteristics of the seeds of P. brutia. Seed dimensions and mean seed fresh and dry weight of trees at a distance 1500 m away from the ring road, were significantly greater than that of seeds from trees were 0, 30, 100 and 500 m away. Huston and Dochinger (1977) reported that the effect of ambient air pollution by SO2 on seed characteristics, had no significant effect in percentages of filled seeds per cone and seed germination capacity of Pinus strobus, while in case of Pinus resinosa the air pollution decreased almost all seed characteristics. Seeds also of Betula papyrifera from elevated CO2 + O3 collected in 2006 had significantly reduced seed mass (Darbah et al., 2008). Palowski (2000) reported that the average number of seeds per one cone of Pinus sylvestris from two polluted and one non-contaminated area, in southern Poland, were similar. Also, our study showed that the moisture content and the embryo length of P. brutia seeds were not affecting the distance from the traffic pollution source. This may 100 90 80 70 60 50 40 30 20 10 0
a b b
c
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Days from the beginning 0m
30m
100m
500m
1500m
Fig. 1. Cumulative germination of P. brutia seeds as affected by the distance from the pollutant source. In 28th day, percentages followed by different letter are significantly different, P ≤ 0.05. Vertical lines represent ±std. error values.
be explained by the fact that tissues covering the embryo of the seed such as endosperm and seed coat, play an important role in protecting the embryo from harmful pollutants (Li et al., 2005). In the present study, the heavy metal analysis showed that of Pb, Cr, Cu, Fe and Mn contents in the seeds were lower than those recorded for other plant tissues collected near to traffic roads (e.g. Sawidis et al., 1995; Chonopoulos et al., 1997; Celik et al., 2005). Pb, Cr and Cu contents in the seeds were not significantly affected by the distance from the ring road, while Fe content was found significantly greater in seeds collected near to the ring road edge (0 m away), and Mn content that was found significantly lower in seeds collected from the longest distance from the road edge (1500 m away). A possible explanation for the lower accumulation of heavy metals in seeds compared with other plant tissues may be the fact that seed is a stage in the plant life cycle that is well protected against various stresses (Li et al., 2005), and especially pine seeds are within the cones, and they are well protected within the bracts of the cones from any external factor. Similarly, Angelova et al. (2003) reported that heavy metals accumulation in seeds of leguminous crops, grown at 0.1 and 15 km distance from the pollution source, was considerably lower than that in roots and leaves. According to Palowski (2000), seeds are probably protected against the poisonous metals due to some physiological barrier protecting organs of generative propagation. However, metal content in plants influenced by several factors such as: pollution intensity, climatic conditions, soil properties, surface structure and age of the tissues and the plants genotype ability to accumulate and dislocate metals (Voutsa et al., 1996; Onder and Dursun, 2006). Even though there were no significant differences in heavy metals content and only few in seed morphological characteristics, related to the distance from the traffic pollution source, great differences were observed in seed physiological characteristics (viability and germination). All seeds collected close to the ring-road (at distances 0 and 30 m from the road edge) were unable to germinate, even those subjected or to cold–moist stratification and mechanical scarification. Thus, based on the physiological behaviour, seeds can be distinguished into two groups, those from trees close to the ring-road that were unable to germinate, and those collected from distances over 100 m from the ring-road, that showed a quite high germination behaviour. However, seeds collected at the distances of 100 and 500 m exhibited significantly lower final germination compared to the seeds of 1500 m distance from the ring-road, which exhibited normal germination behaviour, with germination percentage similar to those reported in other studies for P. brutia (Skordilis and Thanos, 1995; Paitaridou et al., 2006).
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
Previous studies on the effect of air pollutants on seed germination behaviour of other plant species showed similar as well as controversial results. For example, Krishnayya and Beli (1986), found a decrease in the percentage of pollen germination and seed viability in plants growing near highways. Wierzbicka and Obidzinska (1998) who studied the imbibition of seeds in solutions of lead or barium salts, found that the germination was most strongly inhibited in those species of plants whose seed coats were most permeable to lead. The different degree of permeability of seed coats to lead led to a different degree of germination inhibition. On the contrary, Scherbatskoy et al. (1987) reported that seed germination of the species Picea rubens, Abies balsamea, Betula alleghaniensis and Betula papyrifera, under laboratory conditions, did not show significant germination inhibition in response to metal ions (Al, Cu, Pb, Zn). Wong et al. (1984) found also, that there was no significant relationship (P > 0.05) between seed germination of two vegetable crops and the heavy metal concentrations of dust extracts in two studied roadsides of Hong Kong, while the higher metal concentrations caused marked reduction of root growth for both crops. The observed loss of viability and germination ability of the pine seeds, collected near to traffic road, can be explained probably by the strong environmental stress that may affected the pine seed physiology. The reproductive organs (seeds) of the studied pine species, with their complex organization and long duration of the reproductive cycle (approximately 36 months), seems to be very sensitive to the damaging influence of a wide range of anthropogenic contaminants, as reported for other pines (Geras’kin et al., 2005). The long reproductive cycle may provide the opportunities for pollutants to cause genetic changes or other damages which in the initial stages are less obvious than the direct visible effects of pollutants, but in the long term they are more significant. Acknowledgements The authors gratefully acknowledge Dr. Paraskevi Malea for providing laboratory facilities and the two anonymous referees for their valuable comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2011.05.014. References Angelova, V., Ivanova, R., Ivanov, K., 2003. Accumulation of heavy metals in leguminous crops (bean, soybean, peas, lentils and gram). J. Environ. Prot. Ecol. 4, 787–795. Bako, S.P., Funtua, I.I., Ijachi, M., 2005. Heavy metal content of some savanna plant species in relation to air pollution. Water Air Soil Pollut. 161, 125–136. Boydak, M., Dirik, H., Tilki, F., Calikoglu, M., 2003. Effects of water stress on germination in six provenances of Pinus brutia seeds from different bioclimatic zones in Turkey. Turk. J. Agric. For. 27, 91–97. Celik, A., Kartal, A.A., Akdogan, A., Kaska, Y., 2005. Determining the heavy metal pollution in Denizli (Turkey) by using Robinia pseudo-acacia L. Environ. Int. 31, 105–112. Chonopoulos, J., Haidouti, C., Cronopoulou-Sereli, A., Massas, I., 1997. Variations in plant and soil lead and cadmium content in urban parks in Athens, Greece. Sci. Total Environ. 196, 91–98. Darbah, J., Kubiske, M.E., Nelson, N., Oksanen, E., Vapaavuori, E., David, F., Karnosky, D.F., 2008. Effects of decadal exposure to interacting elevated CO2 and/or O3 on paper birch (Betula papyrifera) reproduction. Environ. Pollut. 155, 446–452. De Fré, R., Bruynseraede, P., Kretzschmar, J.G., 1994. Air pollution measurements in traffic tunnels. Environ. Health Perspect. 192, 31–37. Dursun, S., Ozdemir, C., Guc-lu, D., 2002. Chemical treatment of the leather industry wastewater. J. Inst. Sci. Technol. (Gazi Univ.) 15, 451–456.
161
Ewen, C., Anagnostopoulou, M.A., Ward, N.I., 2009. Monitoring of heavy metal levels in roadside dusts of Thessaloniki. Greece in relation to motor vehicle traffic density and flow. Environ. Monit. Assess. 157, 483–498. Farmaki, E.G., Thomaidis, N.S., 2008. Current status of the metal pollution of the environment of Greece – a review. Glob. NEST J. 10, 366–375. Fedorkov, A., 1999. Influence of air pollution on seed quality of Picea obovata. Eur. J. For. Path. 29, 371–375. Fuentes, D., Disante, K.B., Valdecantos, A., Cortina, J., Vallejo, R., 2007. Sensitivity of Mediterranean woody seedlings to copper, nickel and zinc. Chemosphere 66, 412–420. Ganatsas, P., Tsakaldimi, M., 2007. Effect of light conditions and salinity on germination behaviour and early growth of Umbrella pine (Pinus pinea L.) seed. J. Hort. Sci. Biotechnol. 82, 605–610. Ganatsas, P., Tsakaldimi, M., Thanos, C., 2008. Seed and cone diversity and seed germination of Pinus pinea in Strofylia site of the Natura 2000 Network. Biodivers. Conserv. 17, 2427–2439. Geras’kin, S.A., Kim, J.K., Oudalova, A.A., Vasiliyev, D.V., Dikareva, N.S., Zimin, V.L., Dikarev, V.G., 2005. Bio-monitoring the genotoxicity of populations of Scots pine in the vicinity of radioactive storage facility. Mutat. Res. 583, 55–66. Hall, J.L., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 53, 1–11. Huston, D.B., Dochinger, L.S., 1977. Effects of ambient air pollution on cone, seed and pollen characteristics in eastern white and red pines. Environ. Pollut. 12, 1–5. ISTA, 1996. International Rules for Seed Testing. Seed Sci. and Technol. (suppl. 24). Jozic, M., Peer, T., Turk, R., 2009. The impact of the tunnel exhausts in terms of heavy metals to the surrounding ecosystem. Environ. Monit. Assess. 150, 261–271. Korkmaz, A., Korkmaz, Y., 2009. Promotion by 5-aminolevulenic acid of pepper seed germination and seedling emergence under low-temperature stress. Scientia Horticulturae 119, 98–102. Krishnayya, N.S.R., Beli, S.J., 1986. Effect of automobile lead pollution on Cassia tora L. and Cassia occidentalis L. Environ. Pollut. (Ser. A), 221–226. Laschober, C., Limbeck, A., Rendl, J., Puxbaum, H., 2004. Particulate emissions from on-road vehicles in the Kaisermühlen-tunnel (Vienna, Austria). Atmos. Environ. 38, 2187–2195. Lau, O.W., Luk, S.F., 2001. Leaves of Bauhinia blakeana as indicators of atmospheric pollution in Hong Kong. Atmos. Environ. 35, 3113–3120. Li, W., Khan, M.A., Yamaguchi, S., Kamiya, Y., 2005. Effects of heavy metals on seed germination and early growth of Arabidopsis thaliana. Plant Growth Regul. 46, 45–50. Onder, S., Dursun, S., 2006. Air borne heavy metal pollution on Cedrus libani (A. Rich.) in the city centre of Konya (Turkey). Atmos. Environ. 40, 1122–1133. Paitaridou, D., Ganatsas, P., Sotiriou, K., Varvarigos, G., 2006. Research on seed quality of four native pine species. In: Hellenic Forestry Society (Ed.), Proceedings of the 12th National Conference, Drama 2-5 October 2005 , pp. 151–158 (Greek with English summary). Palowski, B., 2000. Seed yield from polluted stands of Pinus sylvestris L. New Forests 20, 15–22. Region of Central Macedonia, 2003. The atmospheric pollution in Thessaloniki city. Department of Environment, Thessaloniki, 36 p. Reyes, O., Mercedes Casal, M., 2002. Effect of high temperatures on cone opening and on the release and viability of Pinus pinaster and P. radiata seeds in NW Spain. Ann. For. Sci. 59, 327–334. Sawidis, T., Marnasidis, A., Zachariadis, G., Stratis, J., 1995. A study of air pollution with heavy metals in Thessaloniki City (Greece) using trees as biological indicators. Arch. Environ. Contam. Toxicol. 28, 118–124. Scherbatskoy, T., Klein, R.M., Badger, G.J., 1987. Germination responses of forest tree seed to acidity and metal ions. Environ. Exp. Bot. 27, 157–164. Skordilis, A., Thanos, C., 1995. Seed stratification and germination strategy in the Mediterranean pines Pinus brutia and P. halepensis. Seed Sci. Res. 5, 151–160. Steffens, J.C., 1990. Heavy metal stress and the phytochelatin response. In: Stress Responses in Plants: Adaptation and Accliminations Mechanisms. Wiley–Liss Inc, pp. 377–394. Stvolinskaya, N.S., 2000. Viability of Taraxacum officinale Wigg. in populations of the city of Moscow in relation to motor transport pollution. Russ. J. Ecol. 31, 129–131. Swaminathan, C., Vinaya Rai, R.S., Suresh, K.K., Sivaganam, K., 1993. Improving seed germination of Debris Indica by vertical sowing. J. Trop. For. Sci. 6, 152–158. Vlachokostas, C., Achillas, C., Moussiopoulos, N., Kalogeropoulos, K., Sigalas, G., Kalognomou, E.A., Banias, G., 2010. Health effects on social costs of particulate and photochemical urban area pollution: a case study for Thessaloniki, Greece. Air Qual. Atmos. Health, doi:10.1007/s11869-010-0096-1. Voutsa, D., Grimanis, A., Samara, C., 1996. Trace elements in vegetables grown in an industrial area in relation to soil and air particulate matter. Environ. Pollut. 94, 325–335. Wierzbicka, M., Obidzinska, J., 1998. The effect of lead on seed imbibition and germination in different plant species. Plant Sci. 137, 155–171. Wong, M.H., Cheung, L.C., Wong, W.C., 1984. Effects of roadside dust on seed germination and root growth of Brassica chinensis and B. parachinensis. Sci. Total Environ. 33, 87–102.
Contents lists available at ScienceDirect
Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot
Effect of air traffic pollution on seed quality characteristics of Pinus brutia Petros Ganatsas a , Marianthi Tsakaldimi a,∗ , Georgios Zachariadis b a b
Laboratory of Silviculture, School of Forestry and Natural Environment, Aristotle University, Thessaloniki 54124, Greece Laboratory of Analytical Chemistry, School of Chemistry, Aristotle University, Thessaloniki 54124, Greece
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 17 May 2011 Accepted 19 May 2011 Keywords: Pine seeds Germination Seed viability Heavy metals Road traffic
a b s t r a c t This study was undertaken to examine to what degree traffic air pollution affect the reproductive material (the seeds) of Pinus brutia. The study focused on the investigation of the seeds morphological characteristics and physiological attitudes as well as heavy metal concentrations of P. brutia trees grown close the ring-road of Thessaloniki (Greece’s second biggest city), polluted by heavy traffic conditions, and how these characteristics are changed with the distance from the pollution source. A sampling procedure was established for seed collection from various distances from the pollution source. Seeds were properly prepared, measured for their morphological characteristics and heavy metal concentrations, as well as for their physiological behaviour (germination and viability). Analysis of the results showed that even though there were no significant differences in seed heavy metal concentrations, and only few in seed morphological characteristics, related to the distance from the traffic pollution source, there was a significant alteration of physiological behaviour for the seeds originated closed to the traffic. All the seed collected close to the ring-road (sample distances 0 and 30 m from the road) were unable to germinate, even those subjected to pre-treatments and repeated germination trials over longer duration, while those collected from distances over 100 m from the ring-road showed a common germination behaviour. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Air pollution affects human health as well as plants and microorganisms. The sampling use of plant tissues has long been shown to be an effective indicator of atmospheric pollution (Sawidis et al., 1995; Dursun et al., 2002). Vehicular traffic emissions play an important role in air pollution. They consist of gaseous pollutants like nitric oxides, carbon monoxide, sulphur dioxide, hydrocarbons, fine and coarse particulate matter like diesel soot and airborne particulate-bound trace metals and metals (De Fré et al., 1994; Laschober et al., 2004). Metallic particles released by motor vehicles may remain in the air for some time, but most of them get deposited on the roadside soils and the plant material close to the road. Metals can enter plants directly via rain and dust but also be taken up from the soil through roots (Jozic et al., 2009). Many trace metals, such as Pb, Zn, Ni, Cd, etc., are released into the atmosphere adjacent to roadways, affecting the roadside habitat (Wong et al., 1984). In recent years, it has been shown that heavy metal levels in soil and vegetation have increased considerably due to traffic pollution and the problem rises as daily traffic increases (Onder and Dursun, 2006). For example, Celik et al. (2005) reported concentration values of Fe, Pb, Cu, Mn in plants at roadsides approximately four times
∗ Corresponding author. E-mail address: [email protected] (M. Tsakaldimi). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.014
greater than values in plants at control sites. A review research published on metal pollution in Greek cities (included Thessaloniki city) reported increased concentrations of the metals Pb, Cu, Zn, Pt and Pd in urban environment such as top-soils of highways and streets. Also, the soil and plant analyses showed that Fe and Cu are the most accumulated elements in plants, while pollution from copper (Cu), iron (Fe) and cadmium (Cd) seems to be the most serious problem (Farmaki and Thomaidis, 2008). However, while some heavy metals perform important biological functions, such as being structural, and catalytic constituents of enzymes and other proteins (Hall, 2002), when in excess heavy metals are toxic to plant cells and may inhibit growth (Steffens, 1990; Hall, 2002). Many of accumulating species (those with resistance), are species with large crowns and broad leaves, although, many conifers have also been found to accumulate heavy metals (Bako et al., 2005). The reproductive organs of coniferous trees, with their complex organization and long duration of the reproductive cycle, are the most sensitive to the damaging influence of a wide range of anthropogenic contaminants (Geras’kin et al., 2005). Especially for pine species with a long maturation period (usually 24–36 months), may provide the opportunities for pollutants genotoxic effects and significant DNA damages in the undifferentiated stem cells, for individuals exposed in situ in polluted areas. Additionally, the presence of mutagenic factors near highways is undoubted; many components of exhaust gases, pavements, and technologic dust may be
158
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
mutagenic (Stvolinskaya, 2000), while mutagenesis is probably not the sole factor responsible for pathological seed development. The seeds as the reproductive organs of seed-bearing plants are especially sensitive to traffic pollutants; thus, in plants growing near highways with heavy traffic, seed viability decreases, and the number of abnormally developing embryos increases more than twice (Stvolinskaya, 2000). Analysing plant tissues can give better results in terms of sensitivity and reproducibility (Lau and Luk, 2001). Uptake of elements into plants can happen via roots from the soil and transported to the leaves and directly via the leaves from the air, or by precipitation. There are many investigations on heavy metal and other pollutant levels in air, and their toxic effect on the plants. Less well known, however, are the effects of chronic exposures on the reproductive capacity of forest trees, particularly in situations where visible injury does not occur. Even though, the effects of air pollutants on foliage of coniferous species are well known (Sawidis et al., 1995; Fuentes et al., 2007; Onder and Dursun, 2006), unfortunately, there are scarce studies dealt with the effect of air pollution on seed ecology of conifers in non-controlled environments. Fedorkov (1999) reported that the viability of Siberian spruce seeds was significantly reduced under severe deposition of sulphur dioxide and heavy metals (nickel and copper) in air. Also, Huston and Dochinger (1977) reported that previous studies on conifer species located in a polluted region or at various distances from cities, noted a marked reduction in cone dimensions, 1000-seed weight and germination capacity. Thessaloniki (Greece’s second biggest city) is considered one of the most polluted cities in Europe, especially with respect to airborne particles (Vlachokostas et al., 2010). The ring road of Thessaloniki separates the city from the sub-urban forest and bears high traffic volume (over 100,000 vehicles daily) with three lanes of traffic per direction. The roadsides characterized by high concentration of air pollutants CO, SO2 , NO2 , PM10 and O3 (Region of Central Macedonia, 2003) and high concentration of heavy metals in roadside dusts (max values of Pb, Cu and Mn 180, 300 and 280 mg/kg, respectively) (Ewen et al., 2009). This study was undertaken to examine to what degree air traffic pollution affect the reproductive organs (the seeds) of Pinus brutia in a non-controlled environment. The study focused on: (a) the investigation of the morphological and physiological characteristics and heavy metal content of seeds of P. brutia trees, grown near to the ring road of Thessaloniki city, and (b) taking into account that the concentration of heavy metals in plants decreases with the distance from the pollution source (Chonopoulos et al., 1997; Celik et al., 2005), how these characteristics are changed with the distance from the ring road.
2. Materials and methods 2.1. Experimental site The ring-road of Thessaloniki city is bordered by the sub-urban forest of the city named “Kedrinos Lofos”, separates the city from the sub-urban forest, and bears large traffic volume. The dominant tree species in the forest is the Mediterranean Pine, P. brutia, which in the great part of the area forms pure, even-aged stands coming from reforestation. The vegetation of the general area belongs to the Quercetalia pubescentis zone and especially to the Ostryo-Carpinion alliance. The soils of the area are slightly acid up to neutral, shallow up to middle depth, poor of nutritious ingredients, with a high percentage of stones and pebbles. The climate is a Mediterranean type with a cool winter and high temperature during the summer. According to meteorological station of Aristotle University of Thessaloniki, which is located close to the study sites, the mean
annually precipitation is 420 mm and the mean annual temperature is 15.6 ◦ C. The dry period lasts from the middle of May to the end of September. 2.2. Cone sampling and preparation of samples Three (3) uniform sampling sites (concerning topography and canopy cover) were chosen for the study, along the ring-road (Suppl. Fig. 1). For each sampling site, cone samples were collected in late Spring 2009 and at five distances from the road edge to the inner part of the forest (0, 30, 100, 500 and 1500 m). At each distance point, five (5) cones were collected from each of 10 selected trees (Reyes and Mercedes Casal, 2002) of P. brutia 50-year-old with similar dimensions (totally 750 cones). None of these trees displayed foliar pollution injury symptoms. All cones were already mature (i.e., brown-coloured). All the collected samples were put in polyethylene bags and transferred to the laboratory. Cone opening was achieved by thermal treatment of cones at 45 ◦ C in an oven, for three days. All seeds were extracted manually. For each sampling site the seeds were pooled for each distance point. Damaged and insect-infected seeds were discarded, and the empty ones were eliminated using the floating method in distilled water (Boydak et al., 2003; Ganatsas et al., 2008). The full filled seeds were stored in a refrigerator at 4 ◦ C. 2.3. Measurements of seed morphological characteristics In order to measure the seed size (length and width), weight and moisture content, 20 full filled seeds from each distance × 3 sampling sites were selected. These measurements were accomplished twice with the same number of sampled seeds. Totally 600 seeds were measured. Their dimensions were measured with callipers in accuracy 0.01 mm. Seeds were weighed with electrical balance of four decimal counters and then they oven-dried at 107 ◦ C for 17 ± 1 h and they weighted again (Paitaridou et al., 2006). The moisture content of seeds was calculated by measuring the weight lost after the seeds oven-drying (ISTA, 1996). The length of embryo (mm) was recorded on a random sample of 30 seeds (10 seeds × 3 sampling sites) from each distance, using a stereoscope (Leica). Photographs were taken on millimetre paper background by a digital photo camera (Nicon BM-7). 2.4. Chemical analysis Chemical analysis of seeds was carried out in order to determine the heavy metals contents in seeds. A sample of pooled seeds of each distance point per sampling site was oven dried at 80 ◦ C for 1 week and was ground to a fine powder by a microhammer mill in order to pass through a 1 mm aperture screen. From each powder sample, three subsamples of 1 g were weighted and, metals were extracted from each subsample by wet digestion method by the use of the acid mixture H2 SO4 + HNO3 + HClO4 . 1 g DW of each subsample was placed in an open quartz tube. Six and half (6.5) ml of the acid mixture (1 ml H2 SO4 + 5 ml HNO3 + 0.5 ml HClO4 ) were added to each tube and the mixture was left 2 h at room temperature. It was warmed for 2 h at 50 ◦ C and then heated at 180 ◦ C for 4 h (Sawidis et al., 1995). Then the solutions were filtered through Whatman type 589/2 filters and the filtrate was diluted to 50 ml volume with extra pure water. Each final solution was put in cleaned plastic containers and analyzed for lead (Pb), chromium (Cr), copper (Cu), iron (Fe) and manganese (Mn) concentrations by Atomic Absorption Spectrometry (Perkin Elmer, 5100 ZL) and Inductively Coupled Plasma Atomic Emission Spectrometry (Perkin Elmer, Optima 3100 XL Plasma Spectrometer). The heavy metals contents are given as g/g dry weight (DW).
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
159
Table 1 Effects of distance, from the ring road edge (pollutant source) to the inner part of the forest, on seed morphological characteristics of P. brutia. Distance from the ring road (m)
Seed length (mm)
Seed width (mm)
Seed fresh weight (g)
Seed dry weight (g)
Seed moisture content (%)
Length of embryo (mm)
0 30 100 500 1500
6.92 (0.26) b 6.88 (0.25) b 6.54 (0.19) b 6.94 (0.15) b 7.66 (0.14) a
3.95 (0.13) b 4.03 (0.09) b 3.96 (0.09) b 4.26 (0.07) ab 4.37 (0.08) a
0.057 (3 × 10−3 ) b 0.055 (3 × 10−3 ) b 0.061 (1 × 10−3 ) b 0.057 (2 × 10−3 ) b 0.070 (3 × 10−3 ) a
0.053 (3 × 10−3 ) b 0.050 (3 × 10−3 ) b 0.057 (1 × 10−3 ) b 0.053 (2 × 10−3 ) b 0.065 (3 × 10−3 ) a
7.20 (0.93) ns 7.36 (0.99) ns 7.63 (0.53) ns 7.49 (0.37) ns 6.23 (0.49) ns
5.8 (0.4) ns 5.8 (0.3) ns 6.0 (0.4) ns 6.2 (0.4) ns 6.5 (0.5) ns
Values of the tested variables are the averages ± the standard error of the mean (in brackets). Within a column, the averages followed by different letter are significantly different, P ≤ 0.05. ns: non significant differences.
2.5. Seed germination and viability experiments 2.5.1. 1st experiment Germination tests were performed in 9-cm-diameter glass petri dishes on two layers of filter paper saturated with distilled water (Ganatsas et al., 2008). Three replications of 25 seeds were used for each distance point and for each sampling site; thus, a total of 225 seeds were used for each distance point (3 replications of 25 seeds × 3 sampling sites). The seeds were immersed in 3% (v/v) formaldehyde/deionized water for 5 min to avoid fungal contamination. After that, the seeds were washed with deionized water. Filter papers were changed every 3 d in order to reduce the chance of cross-contamination by micro-flora (Ganatsas and Tsakaldimi, 2007). Experiments were carried out in a temperature controlled growth chamber at 20 ± 0.5 ◦ C with a photoperiod of 12 h and luminance of 1000 lux (Boydak et al., 2003). Seed germination was measured every 2nd day for 28 d duration, and germination was considered to have occurred if the radical protruded 5 mm from the seed coat. Seeds with abnormal radicals were recorded but they were excluded from the germination counts. Cumulative germination percentage was evaluated every 2 days and the final value was obtained after 28 d. Mean germination time (MGT) and the peak value (PV) were also calculated as these parameters combined with the germination capacity are show better the seed performance. MGT was calculated according to the following A D +A D2 +...AnDn formula:MGT = 1 1A +A2 +...An where A is the number of seeds 1 2 germinating per day, D is the time corresponding to A in days and n is the number of days to final count (Ganatsas et al., 2008; Korkmaz and Korkmaz, 2009). Peak value (PV) is the maximum mean daily germination obtained by dividing the maximum cumulative percentage reached at any time during the test period by the number of days from sowing when that maximum was reached (Swaminathan et al., 1993). 2.5.2. 2nd experiment In the second experiment, new samples of seeds from the sampling distances, that showed null germination, were subjected to cold–moist stratification for two months at a temperature of 2–4 ◦ C in the dark and after that to mechanical scarification with sandpaper (Skordilis and Thanos, 1995). The germination tests were performed as in the previous experiment. At the end of the test, the seeds that were not germinated were subjected to a Tetrazolium test (1% 2,3,5-triphenyl tetrazolium chloride) to determine their viability (ISTA, 1996). If these ungerminated seeds were viable then other reasons would be responsible (e.g. experimental conditions) for germination failure. In the case of the Tetrazolium test, only those seeds that show a significant respiratory activity (dark-red), after 24 h staining period, are considered as viable. 2.6. Statistical analysis √ Percentages data were transformed using the arcsine transformation to meet the assumptions of normality and homoscedasticity. Tables and figures present untransformed data and standard
error of the mean (±SE). One-way ANOVA was used to test differences. Duncan’s test was used to compare the mean values of the studied seed variables among the 5 sampling distances. All statistical analyses were conducted using a critical P value ≤ 0.05. 3. Results 3.1. Seed morphological characteristics Seed overall morphological characteristics are shown in Table 1. Seed dimensions of trees from the inner part of the forest (at 1500 m from the ring road edge), were significantly greater (7.66 mm length and 4.37 mm width) than that of the trees were closer to the ring-road edge. Seed dimensions of trees were 0, 30, 100 and 500 m away from the ring-road, did not show any significant differences. The mean seed fresh and dry weight of the studied seeds was fluctuated from 0.057 to 0.070 g and from 0.050 to 0.065 g respectively, while the significantly greatest values were observed in seeds of trees abstained 1500 m from the ring-road. However, between the studied seeds, non-significant differences were observed in seed moisture content that ranged from 6.23 to 7.63%. The length of seed embryo was ranged from 5.5 to 6.5 mm and was slightly greater in the seeds at the distances 500 and 1500 m, but no significant differences were detected. 3.2. Seed heavy metal content The obtained results from the heavy metal analysis in the studied seeds of P. brutia are given in Table 2. The heavy metals content in seeds, collected at various distances from the ring-road edge to the inner part of the sub-urban forest, generally did not show significant differences, with few exceptions. Fe content was found significantly greatest in seeds of trees near to the ring-road edge (0 m away), while Mn content was found significantly lowest in seeds collected from the trees existed in the longest distance from the road edge (1500 m away). However, Pb content of the seeds was found always lower than the detection limit of 1 g/g DW. 3.3. Seed germination behaviour and viability According to the results from the first experiment, there was a significant reduction in seed germination of P. brutia as the distance from the ring-road decreased (Fig. 1). The highest seed germination capacity (84%) was observed in trees located 1500 m away from the ring-road (towards the heart of the sub-urban forest). There were no significant differences on seed germination capacity between the trees located at 100 and 500 m away from the ring road; their seed germination was fluctuated between 61 and 63%. Between the above three distances there was no variation in time taken for the initiation of seed germination (germination started 12 days after sowing) and no significant differences were observed for the PV and MGT (Fig. 1, Table 3). However, at distances 0 and 30 m, the seed germination was completely inhibited and no seeds germinated.
160
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
Table 2 Content of Pb, Cr, Cu, Fe and Mn (g/g DW) in seeds of P. brutia. Distance from ring road edge (m)
Pb
Cr
Cu
Fe
Mn
0 30 100 500 1500
<1 <1 <1 <1 <1
1.467 (0.89) ns 0.923 (0.13) ns 1.006 (0.13) ns 0.984 (0.14) ns 1.230 (0.22) ns
11.21 (1.04) ns 9.60 (0.58) ns 8.50 (1.09) ns 8.36 (0.43) ns 10.12 (0.44) ns
135.05 (15.9) a 108.86 (2.2) b 94.38 (4.2) b 95.17 (5.2) b 108.95 (12.3) b
33.72 (2.80) a 36.83 (1.63) a 37.78 (1.21) a 34.93 (2.29) a 22.68 (1.43) b
Values are the averages (g/g DW) from 9 replications ± the standard error of the mean (in brackets). Within a column, the averages followed by different letter are significantly different, P ≤ 0.05. ns: non significant differences. Table 3 Effect of distance, from the ring road (pollutant source) to the inner part of the forest, on mean germination time (MGT, days) and peak value (PV) of P. brutia seeds. Distance from the ring road (m)
MGT PV
0
30
100
500
1500
0.0 0.0
0.0 0.0
23.8 (2.8) ns 2.18 (0.43) ns
24.4 (2.9) ns 2.24 (0.41) ns
24.0 (2.5) ns 3.00 (0.59) ns
Values are the mean and the standard error of the mean (in brackets). ns: non significant differences, P > 0.05.
The results from the second experiment, studying the seeds from the sampling distances 0 and 30 m from the ring-road, which subjected previously to cold–moist stratification and to mechanical scarification, confirmed that the germination capacity of these seeds was null. Similarly, the tetrazolium test revealed that these seeds were not viable at all, after 24 h staining period. 4. Discussion
Germination %
According to the results analysis, the distance from the traffic pollution source (ring-road of Thessaloniki city) significantly affected some morphological characteristics of the seeds of P. brutia. Seed dimensions and mean seed fresh and dry weight of trees at a distance 1500 m away from the ring road, were significantly greater than that of seeds from trees were 0, 30, 100 and 500 m away. Huston and Dochinger (1977) reported that the effect of ambient air pollution by SO2 on seed characteristics, had no significant effect in percentages of filled seeds per cone and seed germination capacity of Pinus strobus, while in case of Pinus resinosa the air pollution decreased almost all seed characteristics. Seeds also of Betula papyrifera from elevated CO2 + O3 collected in 2006 had significantly reduced seed mass (Darbah et al., 2008). Palowski (2000) reported that the average number of seeds per one cone of Pinus sylvestris from two polluted and one non-contaminated area, in southern Poland, were similar. Also, our study showed that the moisture content and the embryo length of P. brutia seeds were not affecting the distance from the traffic pollution source. This may 100 90 80 70 60 50 40 30 20 10 0
a b b
c
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Days from the beginning 0m
30m
100m
500m
1500m
Fig. 1. Cumulative germination of P. brutia seeds as affected by the distance from the pollutant source. In 28th day, percentages followed by different letter are significantly different, P ≤ 0.05. Vertical lines represent ±std. error values.
be explained by the fact that tissues covering the embryo of the seed such as endosperm and seed coat, play an important role in protecting the embryo from harmful pollutants (Li et al., 2005). In the present study, the heavy metal analysis showed that of Pb, Cr, Cu, Fe and Mn contents in the seeds were lower than those recorded for other plant tissues collected near to traffic roads (e.g. Sawidis et al., 1995; Chonopoulos et al., 1997; Celik et al., 2005). Pb, Cr and Cu contents in the seeds were not significantly affected by the distance from the ring road, while Fe content was found significantly greater in seeds collected near to the ring road edge (0 m away), and Mn content that was found significantly lower in seeds collected from the longest distance from the road edge (1500 m away). A possible explanation for the lower accumulation of heavy metals in seeds compared with other plant tissues may be the fact that seed is a stage in the plant life cycle that is well protected against various stresses (Li et al., 2005), and especially pine seeds are within the cones, and they are well protected within the bracts of the cones from any external factor. Similarly, Angelova et al. (2003) reported that heavy metals accumulation in seeds of leguminous crops, grown at 0.1 and 15 km distance from the pollution source, was considerably lower than that in roots and leaves. According to Palowski (2000), seeds are probably protected against the poisonous metals due to some physiological barrier protecting organs of generative propagation. However, metal content in plants influenced by several factors such as: pollution intensity, climatic conditions, soil properties, surface structure and age of the tissues and the plants genotype ability to accumulate and dislocate metals (Voutsa et al., 1996; Onder and Dursun, 2006). Even though there were no significant differences in heavy metals content and only few in seed morphological characteristics, related to the distance from the traffic pollution source, great differences were observed in seed physiological characteristics (viability and germination). All seeds collected close to the ring-road (at distances 0 and 30 m from the road edge) were unable to germinate, even those subjected or to cold–moist stratification and mechanical scarification. Thus, based on the physiological behaviour, seeds can be distinguished into two groups, those from trees close to the ring-road that were unable to germinate, and those collected from distances over 100 m from the ring-road, that showed a quite high germination behaviour. However, seeds collected at the distances of 100 and 500 m exhibited significantly lower final germination compared to the seeds of 1500 m distance from the ring-road, which exhibited normal germination behaviour, with germination percentage similar to those reported in other studies for P. brutia (Skordilis and Thanos, 1995; Paitaridou et al., 2006).
P. Ganatsas et al. / Environmental and Experimental Botany 74 (2011) 157–161
Previous studies on the effect of air pollutants on seed germination behaviour of other plant species showed similar as well as controversial results. For example, Krishnayya and Beli (1986), found a decrease in the percentage of pollen germination and seed viability in plants growing near highways. Wierzbicka and Obidzinska (1998) who studied the imbibition of seeds in solutions of lead or barium salts, found that the germination was most strongly inhibited in those species of plants whose seed coats were most permeable to lead. The different degree of permeability of seed coats to lead led to a different degree of germination inhibition. On the contrary, Scherbatskoy et al. (1987) reported that seed germination of the species Picea rubens, Abies balsamea, Betula alleghaniensis and Betula papyrifera, under laboratory conditions, did not show significant germination inhibition in response to metal ions (Al, Cu, Pb, Zn). Wong et al. (1984) found also, that there was no significant relationship (P > 0.05) between seed germination of two vegetable crops and the heavy metal concentrations of dust extracts in two studied roadsides of Hong Kong, while the higher metal concentrations caused marked reduction of root growth for both crops. The observed loss of viability and germination ability of the pine seeds, collected near to traffic road, can be explained probably by the strong environmental stress that may affected the pine seed physiology. The reproductive organs (seeds) of the studied pine species, with their complex organization and long duration of the reproductive cycle (approximately 36 months), seems to be very sensitive to the damaging influence of a wide range of anthropogenic contaminants, as reported for other pines (Geras’kin et al., 2005). The long reproductive cycle may provide the opportunities for pollutants to cause genetic changes or other damages which in the initial stages are less obvious than the direct visible effects of pollutants, but in the long term they are more significant. Acknowledgements The authors gratefully acknowledge Dr. Paraskevi Malea for providing laboratory facilities and the two anonymous referees for their valuable comments. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2011.05.014. References Angelova, V., Ivanova, R., Ivanov, K., 2003. Accumulation of heavy metals in leguminous crops (bean, soybean, peas, lentils and gram). J. Environ. Prot. Ecol. 4, 787–795. Bako, S.P., Funtua, I.I., Ijachi, M., 2005. Heavy metal content of some savanna plant species in relation to air pollution. Water Air Soil Pollut. 161, 125–136. Boydak, M., Dirik, H., Tilki, F., Calikoglu, M., 2003. Effects of water stress on germination in six provenances of Pinus brutia seeds from different bioclimatic zones in Turkey. Turk. J. Agric. For. 27, 91–97. Celik, A., Kartal, A.A., Akdogan, A., Kaska, Y., 2005. Determining the heavy metal pollution in Denizli (Turkey) by using Robinia pseudo-acacia L. Environ. Int. 31, 105–112. Chonopoulos, J., Haidouti, C., Cronopoulou-Sereli, A., Massas, I., 1997. Variations in plant and soil lead and cadmium content in urban parks in Athens, Greece. Sci. Total Environ. 196, 91–98. Darbah, J., Kubiske, M.E., Nelson, N., Oksanen, E., Vapaavuori, E., David, F., Karnosky, D.F., 2008. Effects of decadal exposure to interacting elevated CO2 and/or O3 on paper birch (Betula papyrifera) reproduction. Environ. Pollut. 155, 446–452. De Fré, R., Bruynseraede, P., Kretzschmar, J.G., 1994. Air pollution measurements in traffic tunnels. Environ. Health Perspect. 192, 31–37. Dursun, S., Ozdemir, C., Guc-lu, D., 2002. Chemical treatment of the leather industry wastewater. J. Inst. Sci. Technol. (Gazi Univ.) 15, 451–456.
161
Ewen, C., Anagnostopoulou, M.A., Ward, N.I., 2009. Monitoring of heavy metal levels in roadside dusts of Thessaloniki. Greece in relation to motor vehicle traffic density and flow. Environ. Monit. Assess. 157, 483–498. Farmaki, E.G., Thomaidis, N.S., 2008. Current status of the metal pollution of the environment of Greece – a review. Glob. NEST J. 10, 366–375. Fedorkov, A., 1999. Influence of air pollution on seed quality of Picea obovata. Eur. J. For. Path. 29, 371–375. Fuentes, D., Disante, K.B., Valdecantos, A., Cortina, J., Vallejo, R., 2007. Sensitivity of Mediterranean woody seedlings to copper, nickel and zinc. Chemosphere 66, 412–420. Ganatsas, P., Tsakaldimi, M., 2007. Effect of light conditions and salinity on germination behaviour and early growth of Umbrella pine (Pinus pinea L.) seed. J. Hort. Sci. Biotechnol. 82, 605–610. Ganatsas, P., Tsakaldimi, M., Thanos, C., 2008. Seed and cone diversity and seed germination of Pinus pinea in Strofylia site of the Natura 2000 Network. Biodivers. Conserv. 17, 2427–2439. Geras’kin, S.A., Kim, J.K., Oudalova, A.A., Vasiliyev, D.V., Dikareva, N.S., Zimin, V.L., Dikarev, V.G., 2005. Bio-monitoring the genotoxicity of populations of Scots pine in the vicinity of radioactive storage facility. Mutat. Res. 583, 55–66. Hall, J.L., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 53, 1–11. Huston, D.B., Dochinger, L.S., 1977. Effects of ambient air pollution on cone, seed and pollen characteristics in eastern white and red pines. Environ. Pollut. 12, 1–5. ISTA, 1996. International Rules for Seed Testing. Seed Sci. and Technol. (suppl. 24). Jozic, M., Peer, T., Turk, R., 2009. The impact of the tunnel exhausts in terms of heavy metals to the surrounding ecosystem. Environ. Monit. Assess. 150, 261–271. Korkmaz, A., Korkmaz, Y., 2009. Promotion by 5-aminolevulenic acid of pepper seed germination and seedling emergence under low-temperature stress. Scientia Horticulturae 119, 98–102. Krishnayya, N.S.R., Beli, S.J., 1986. Effect of automobile lead pollution on Cassia tora L. and Cassia occidentalis L. Environ. Pollut. (Ser. A), 221–226. Laschober, C., Limbeck, A., Rendl, J., Puxbaum, H., 2004. Particulate emissions from on-road vehicles in the Kaisermühlen-tunnel (Vienna, Austria). Atmos. Environ. 38, 2187–2195. Lau, O.W., Luk, S.F., 2001. Leaves of Bauhinia blakeana as indicators of atmospheric pollution in Hong Kong. Atmos. Environ. 35, 3113–3120. Li, W., Khan, M.A., Yamaguchi, S., Kamiya, Y., 2005. Effects of heavy metals on seed germination and early growth of Arabidopsis thaliana. Plant Growth Regul. 46, 45–50. Onder, S., Dursun, S., 2006. Air borne heavy metal pollution on Cedrus libani (A. Rich.) in the city centre of Konya (Turkey). Atmos. Environ. 40, 1122–1133. Paitaridou, D., Ganatsas, P., Sotiriou, K., Varvarigos, G., 2006. Research on seed quality of four native pine species. In: Hellenic Forestry Society (Ed.), Proceedings of the 12th National Conference, Drama 2-5 October 2005 , pp. 151–158 (Greek with English summary). Palowski, B., 2000. Seed yield from polluted stands of Pinus sylvestris L. New Forests 20, 15–22. Region of Central Macedonia, 2003. The atmospheric pollution in Thessaloniki city. Department of Environment, Thessaloniki, 36 p. Reyes, O., Mercedes Casal, M., 2002. Effect of high temperatures on cone opening and on the release and viability of Pinus pinaster and P. radiata seeds in NW Spain. Ann. For. Sci. 59, 327–334. Sawidis, T., Marnasidis, A., Zachariadis, G., Stratis, J., 1995. A study of air pollution with heavy metals in Thessaloniki City (Greece) using trees as biological indicators. Arch. Environ. Contam. Toxicol. 28, 118–124. Scherbatskoy, T., Klein, R.M., Badger, G.J., 1987. Germination responses of forest tree seed to acidity and metal ions. Environ. Exp. Bot. 27, 157–164. Skordilis, A., Thanos, C., 1995. Seed stratification and germination strategy in the Mediterranean pines Pinus brutia and P. halepensis. Seed Sci. Res. 5, 151–160. Steffens, J.C., 1990. Heavy metal stress and the phytochelatin response. In: Stress Responses in Plants: Adaptation and Accliminations Mechanisms. Wiley–Liss Inc, pp. 377–394. Stvolinskaya, N.S., 2000. Viability of Taraxacum officinale Wigg. in populations of the city of Moscow in relation to motor transport pollution. Russ. J. Ecol. 31, 129–131. Swaminathan, C., Vinaya Rai, R.S., Suresh, K.K., Sivaganam, K., 1993. Improving seed germination of Debris Indica by vertical sowing. J. Trop. For. Sci. 6, 152–158. Vlachokostas, C., Achillas, C., Moussiopoulos, N., Kalogeropoulos, K., Sigalas, G., Kalognomou, E.A., Banias, G., 2010. Health effects on social costs of particulate and photochemical urban area pollution: a case study for Thessaloniki, Greece. Air Qual. Atmos. Health, doi:10.1007/s11869-010-0096-1. Voutsa, D., Grimanis, A., Samara, C., 1996. Trace elements in vegetables grown in an industrial area in relation to soil and air particulate matter. Environ. Pollut. 94, 325–335. Wierzbicka, M., Obidzinska, J., 1998. The effect of lead on seed imbibition and germination in different plant species. Plant Sci. 137, 155–171. Wong, M.H., Cheung, L.C., Wong, W.C., 1984. Effects of roadside dust on seed germination and root growth of Brassica chinensis and B. parachinensis. Sci. Total Environ. 33, 87–102.