Assessment of both environmental cytotoxicity and trace metalpollution using Populus simonii Carr. as a bioindicator

Victor Sluchyk & Iryna Sluchyk & Alexander Shyichuk

Received: 9 January 2014 /Accepted: 11 June 2014# Springer International Publishing Switzerland 2014

Abstract The level of environmental pollution in thecity of Ivano-Frankivsk (Western Ukraine) has beenassessed by means of roadside poplar trees asbioindicators. Dividable apical meristem cells of rudi-mentary leaves were quantitatively analysed for mitoticactivity and distribution. Anaphases were further exam-ined for chromosomal aberrations. Male catkins werealso examined for sterile pollens. Accumulation of traceelements in vegetative buds was also evaluated in orderto reveal source(s) of environmental pollution. Poplartrees growing in the urban environment proved to haveincreased chromosomal aberrations (up to 4-fold) andincreased pollen sterility (up to 4-fold) as well as de-creased mitotic activity (by factor 1.5) as compared tocontrol sampling site. The biomarker data correlatemoderately with increased (up to 4-fold) concentrationsof Ni, Zn, Pb, Cd and Cu in vegetative tissues suggest-ing that probable cause of the environmental cytotoxic-ity may be vehicle emissions. The maximum increase inchromosomal aberrations (7-fold) and the minimummitotic activity (half of the control one) were recordedin poplar trees growing in industrial suburb in vicinity of

large cement production plant. Taking in mind insignif-icant bioaccumulation of trace elements in the industrialsuburb, the high environmental toxicity has been as-cribed to contamination in cement and asbestosparticulates.

Keywords Bioindication . Urban environment .

Karyokinesis pathology. Pollen viability .

SEM-EDXMA

Introduction

Bioindicator plants are frequently used to indicate tracemetal pollutions in the environment. In particular, spe-cies within the genus Populus are well known to beeffective phytoabsorbers of trace elements (Laureysenset al. 2004; Castiglione et al. 2009; Vollenweider et al.2011; Van Nevel et al. 2011; Jun and Ling 2012) owingto their widespread root system and large water uptake.It is also favourable that long-living poplar trees arewidely used in land management as both decorativeplants and windbreaks. For these reasons, poplar provedto be suitable bioindicator to assess cumulative level ofenvironmental pollution with trace elements. For exam-ple, Lombardy poplar (Populus nigra) has been used asstandardized bioindicator plant for mapping and long-time monitoring of heavy metal pollutions in Bulgaria(Djingova et al. 1995, 1996, 1999, 2001). Leaves, stemsand fruiting catkins of white poplar (Populus alba) havebeen used for long-time monitoring of soil pollutionwith As, Cd, Cu, Fe, Mn, Ni, Pb and Zn in a Southern

Environ Monit AssessDOI 10.1007/s10661-014-3879-2

V. Sluchyk : I. SluchykDepartment of Biology and Ecology, Vasyl’ StefanykPrecarpathian National University,Shevchenka 57, Ivano-Frankivsk 76000, Ukraine

I. Sluchyke-mail: sluchyk@optima.com.ua

A. Shyichuk (*)University of Technology and Life Sciences,Seminaryjna 3, 85-326 Bydgoszcz, Polande-mail: szyjczuk@utp.edu.pl

Spain region affected by a mine spill (Madejón et al.2004, 2013). Positive correlation between traffic densityand trace element content in leaves and bark of Carolinapoplar (Populus×canadensisMoench) has been report-ed by Celik et al. (2010). Due to good bioaccumulation,poplar leaves proved to be well suited to indicate traceelements in soil at concentrations less than detectionlimits of ICP-AES technique (Madejón et al. 2013).

On the other hand, overall anthropogenic pollution ofthe environment may be indicated by means of cytoge-netic markers such as chromosomal aberrations, mitosisdisturbances, micronuclei frequency and pollen viability(Fargašová 2012; Geras’kin et al. 2011; Malayeri et al.2012). The most often used bioindicator plants are var-ious grassy species of genera Tradescantia, Vicia, Alli-um, etc. while woody species are rarely used (Prus-Głowacki et al. 2006; Belousov et al. 2012).

The objective of this study was to assess pollution ofthe urban area using vegetative tissues of Populussimonii Carr. Mitotic activity, chromosomal aberrationsand pollen sterility were used as biomarkers. Bioaccu-mulation of trace elements in vegetative tissues was alsoevaluated in order to reveal sources of the environmentalpollution.

Materials and methods

Study area

The study was carried out in Ivano-Frankivsk, a city insouthwestern Ukraine. Its population is 260,000 inhab-itants and the area is 93 km2 (with 60 % residential,15 % municipal and 25 % industrial area). The cityhouses approximately 60 small factories (mainly ofmechanical engineering profile) and has intensive transittraffic. Five sampling sites (denoted here as North,Northeast, Central, South, Southeast) were located with-in the city border. Onemore sampling site was located inindustrial suburb (village Yamnytsa) in the vicinity of alarge cement and fibre cement plant. Control samplingsite has been located in the town of Rozhniativ whichhas very similar soil and climatic conditions as well asfairly low industrial and traffic activity.

Vegetative samples

Five trees of P. simonii Carr. of the same age (60–80 years) were chosen in each of the sampling sites.

Distances of the trees from a main road did notexceed 10 m. Third to fifth vegetative buds from1-year shoots were taken in the period of vernalplant sap movement from the top leeward(northwest) side of each selected tree. Male catkinswere sampled during mass flowering period from 1-year shoots on the top leeward side of each selectedtree. The obtained biomaterial was fixed in Carnoy’smixture at 0–4 °C for 24 h.

Bioassays

Meristem cells from rudimentary leaves were stained in4 % aceto-iron-haematoxylin. Subsequent clarification,preservation and preparation of the squash slides wereperformed in the Hoyer’s fixative solution. The micro-images were obtained using the Olympus CX-300 mi-croscope equipped with the Olympus SP-500 UZ cam-era. Image analysis was performed using the QuickPHOTO MICRO 2.3 for Windows software(Olympus). At least 1,000 dividable cells of each treewere quantitatively analysed for mitotic activity anddistribution. At least 300 anaphases of each tree werequantitatively analysed for chromosome abnormalities(fragments, laggards and bridges). Determination ofsterile pollens was carried out using the acetocarminestaining method (Rotreklová 2008; Malayeri et al.2012). Numbers of the analysed pollens were 600 to1,000 for each tree. The obtained percentages of sterilepollens were statistically evaluated by means of theStudent’s t test (P<0.05).

Energy-dispersive X-ray microanalysis(SEM-EDXMA)

The sampled vegetative buds were washed with EDTAsolution, rinsed with de-ionised water and incinerated ina quartz crucible. The obtained ash samples werepressed into tablets, glued on copper pads, coated bycarbon layer and subjected to electron beam in thescanning electron microscope REMMA-102 Eequipped for energy-dispersive X-ray spectroscopy.Concentrations of trace elements of interest were deter-mined using calibration samples prepared by means ofthe standard addition method using analytical gradenitrates and sulphates of Cd, Pb, Mn, Cr, Ni, Zn, Cu,Al and Fe.

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Results

Assessment of environmental toxicity using cytogeneticmarkers

Level of environmental cytotoxicity was evaluatedusing both somatic cells and gametes of poplar trees(Table 1, Figs. 1, 2 and 3). Numbers of chromosomalaberrations in living meristem cells proved to be depen-dent on the sampling site (Table 1). At first, no statisti-cally significant differences in the average number ofaberrations per single cell were found in the South andSoutheast sites. On the other hand, significant numbersof chromosomal aberrations were registered in North,Northeast and Central sites (Table 1). The maximalincrease in chromosomal aberrations (6.8-fold as com-pared to the control site) was registered in the Industrialsuburb, indicating clearly that pollution level exceededthe buffering capacity of the local ecosystem. Mitosischaracteristics also proved to be dependent on samplingsite (Figs. 1 and 2). Mitotic activity was found to besubstantially decreased in the North and Northeast siteswhile the least value of mitotic index (half of the controlone) has been registered in the Industrial suburb (Fig. 1).Distribution of cells on mitotic phases proved to beunchanged in the South and Southeast sites (the dataare not shown). In the rest of the sampling sites, thisdistribution proved to be markedly distorted with sig-nificant decrease in prophase cell fraction as well as withincrease in metaphase and telophase ones (Fig. 2). In allthe polluted sites (except Northeast) the metaphase in-dex exceeded the prophase one (Fig. 2) indicating that

mitotic division is the only process occurring. Verysimilar inference on environmental pollution was madeby means of sterile pollen percentages—increased envi-ronmental toxicity was registered in the North, North-east and Central sites as well as in the Industrial suburb(Fig. 3).

Assessment of trace element pollutions usingbioaccumulation data

Evaluation of trace elements in vegetative tissues wasperformed having in mind that metal ions are wide-spread pollutants in urban areas. High ability of poplarleaves for trace element bioaccumulation wasfavourable to overcome rather low sensitivity of theanalytical technique used in this study. Concentrationsof Ni, Zn, Cu, Cd and Pb in the urban tree tissues provedto be significantly increased as compared to the controlones (Table 2) while concentrations of other trace ele-ments (Cr, Al, Fe, Mn) remain close to the control ones

Table 1 Level of chromosomal aberrations in meristem cells ofpoplar trees

Sampling site Number of anaphases Percentage ofanaphases withaberrationsTotal

anaphasesAnaphases withrearrangements

Control 1539 16 1.04±0.07

Southeast 1519 18 1.18±0.17

South 1518 21 1.39±0.20

Northeast 1536 30 1.95±0.23a

North 1545 38 2.46±0.39a

Central 1552 64 4.13±0.41a

Industrial suburb 1519 107 7.04±0.70a

a Significant differences in comparison with the control

MitIndex

control 118

Southeast 111

South 102

Northeast 84

North 76

Central 109

Industrial suburb 56

0

20

40

60

80

100

120

‰

Fig. 1 Mitotic index of poplar tree meristem cells

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(the data are not shown). The most polluted tissuesproved to be those sampled from the North, Northeastand Central sites (Table 2). The factors of trace elementcontent increase (1.8 to 4) were similar to those (ap-proximately 2) that have been observed by Djingovaet al. (1999) in Bulgarian cities having comparablepopulation. The largest increase in tissue concentrationswas registered for nickel and zinc (Table 2). This factagrees well with numerous literature reports suggestingthat zinc is easily accumulated and translocated frompoplar roots to leaves and twigs (Laureysens et al. 2004;Castiglione et al. 2009; Lettens et al. 2011).

prophase metaphase anaphase telophase

control 45 26.5 5.3 20

Northeast 36 32 5.2 25.5

North 32.5 36.5 5 25.5

Central 29.5 42 5.2 23

Industrial s 28 37 5.1 28

0

10

20

30

40

50

prophase metaphase anaphase telophase

% Northeast

North

Central

Industrial suburb

control

Fig. 2 Distribution of meristemcells on mitotic phases

sterile

control 6.2

Southeast 6.6

South 7.6

Northeast 12

North 14.3

Central 25.3

Industrial suburb 18.5

0

5

10

15

20

25

%

Fig. 3 Percentages of sterile pollens in poplar trees

Table 2 Concentrations of trace elements in vegetative tissues ofpoplar trees, (μg/g)

Sampling site Cu Cd Pb Zn Ni

Control 3.3 0.17 1.6 42 0.85

Southeast 5.6 0.36 3.5 90 2.2

South 8.9 0.34 3 67 2.4

Northeast 10.1 0.4 4.8 163 3

North 8.2 0.42 3.2 151 2.2

Central 5.9 0.31 5.1 166 3.4

Industrial suburb 5.3 0.25 1.4 43 0.76

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Discussion

The highest chromosomal damage and the lowest mi-totic activity were registered in the Industrial suburb(Table 1, Fig. 1). This fact indicates clearly that theIndustrial suburb is the site that suffers from the highestenvironmental toxicity. This conclusion is confirmedalso with rather high percentage of sterile pollens(Fig. 3) which resulted from meiosis disturbances. Theobserved high level of environmental toxicity cannot beassociated with metal pollutants since trace elementconcentrations in vegetative tissues remain close tothose registered in the control non-contaminated site(Table 2). The most probable cause of the high environ-mental cytotoxicity is pollutant emission from the ce-ment plant located in the Industrial suburb. According toreports of the regional agency of environmental protec-tion, total annual atmospheric emission in the Industrialsuburb exceeds almost triple the sum of total emissionsin the city (110,000 vs. 40,000 t, respectively). Alkalinefly ash and cement dust are well known to cause multi-ple adverse effects in exposed trees (Kask et al. 2008;Dziri and Hosni 2012; Paal et al. 2013). Chrysotileasbestos fibres were reported to be as well toxic forplants (Trivedi and Ahmad 2011).

To discuss probable cause of environmental toxicityin the rest of the sampling sites, data on trace elementbioaccumulation should be taken into consideration.The obtained biomarker data correlate moderately withtrace element concentrations registered in the Central,North, Northeast, South, Southeast and control sites(Table 3). In particular, chromosomal damage and pol-len sterility correlate well with Pb, Zn and Ni concen-trations while Mitotic Index correlates with Cu and Cdones (Table 3). Taking into account that no large indus-tries are located in the sampling sites, the increased traceelement concentrations may be associated with vehicle

emissions. Roadside soils and plants are well known tobe enriched with heavy elements, mainly Cd, Zn, Pb, Niand Cu (Swaileh et al. 2006; Celik et al. 2010;Princewill-Ogbonna and Ogbonna 2011; Kluge andWessolek 2012; Nazzal et al. 2013). An additional con-firmation is the fact that no marked accumulation oftrace elements was observed in the Industrial suburb(Table 2) having relatively low automotive traffic. Onecan summarize that the high environmental cytotoxicityin the North, Northeast and Central sites has resultedfrom traffic-related metal contaminations. This conclu-sion agrees well with literature reports on antimitoticand genotoxic effects in plants caused by heavy ele-ments (Pourrut et al. 2011; Fargašová 2012; Belousovet al. 2012). Intrinsic toxicity of elements of changeablevalence is probably related with initiation of free radicalchain reactions. The chain reaction intermediates aresuperoxide and hydroxyl radicals resulting in oxidativedamage of membrane lipids, proteins and DNA(Lushchak et al. 2008; Sun et al. 2010; Kubrak et al.2010; Bhaduri and Fulekar 2012; Sytar et al. 2013).

Conclusions

Poplar trees proved to be very useful bioindicators toassess pollution of the urban area. Overall environmen-tal cytotoxicity was assessed via measuring chromo-somal aberrations and mitosis abnormalities in meristemcells of rudimentary leaves. The percentage of sterilepollens was an additional measure of the environmentalpollution. Data on bioaccumulation of trace elements inpoplar leaves were useful to reveal probable sources ofenvironmental toxicity.

References

Belousov, M. V., Mashkina, O. S., & Popov, V. N. (2012).Cytogenetic response of Scots pine (Pinus sylvestrisLinnaeus, 1753) (Pinaceae) to heavy metals. ComparativeCytogenetics, 6, 93–106.

Bhaduri, A. M., & Fulekar, M. H. (2012). Antioxidant enzymeresponses of plants to heavy metal stress. Reviews inEnvironmental Science and Biotechnology, 11, 55–69.

Castiglione, S., Todeschini, V., Franchin, C., Torrigiani, P.,Gastaldi, D., Cicatelli, A., et al. (2009). Clonal differencesin survival capacity, copper and zinc accumulation, and cor-relation with leaf polyamine levels in poplar: a large-scalefield trial on heavily polluted soil. Environmental Pollution,157, 2108–2117.

Table 3 Correlation coefficients between biomarkers and traceelement concentrations in the Central, North, Northeast, South,Southeast and control sampling sites

Cu Cd Pb Zn Ni

Anaphaseaberrations

0.109 0.227 0.719 0.78 0.724

Sterile pollencontent

0.096 0.204 0.726 0.784 0.721

Mitotic index −0.804 −0.812 −0.326 −0.645 −0.359

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Celik, S., Yucel, E., Celik, S., Gucel, S., & Ozturk, M. (2010).Carolina poplar (Populus×canadensisMoench) as a biomon-itor of trace elements in Black sea region of Turkey. Journalof Environmental Biology, 31(1–2), 225–232.

Djingova, R., Wagner, G., & Peshev, D. (1995). Heavy metaldistribution in Bulgaria using Populus nigra ‘Italica’ as abiomonitor. Science of the Total Environment, 172, 151–158.

Djingova, R., Wagner, G., Kuleff, I., & Peshev, D. (1996).Investigations on the time-dependant variations in metal con-centration in the leaves of Populus nigra ‘Italica’. Science ofthe Total Environment, 184, 197–202.

Djingova, R., Wagner, G., & Kuleff, I. (1999). Screening of heavymetal pollution in Bulgaria using Populus nigra ‘Italica’.Science of the Total Environment, 234, 175–184.

Djingova, R., Ivanova, J., Wagner, G., Korhammer, S., &Markert,B. (2001). Distribution of lanthanoids, Be, Bi, Ga, Te, Tl, Thand U on the territory of Bulgaria using Populus nigra‘Italica’ as an indicator. Science of the Total Environment,280, 85–91.

Dziri, S., & Hosni, K. (2012). Effects of cement dust on volatile oilconstituents and antioxidative metabolism of Aleppo pine(Pinus halepensis) needles. Acta Physiologiae Plantarum,34, 1669–1678.

Fargašová, A. (2012). Plants as models for chromium and nickelrisk assessment. Ecotoxicology, 21, 1476–1483.

Geras’kin, S., Oudalova, A., Michalik, B., Dikareva, N., &Dikarev, V. (2011). Geno-toxicity assay of sediment andwater samples from the Upper Silesia post-mining areas,Poland by means of Allium-test. Chemosphere, 83, 1133–1146.

Jun, R., & Ling, T. (2012). Increase of Cd accumulation in fivepoplar (Populus L.) with different supply levels of Cd.International Journal of Phytoremediation, 14, 101–113.

Kask, R., Ots, K., Mandre, M., & Pikk, J. (2008). Scots pine(Pinus sylvestris L.) wood properties in an alkaline air pollu-tion environment. Trees, 22, 815–823.

Kluge, B., &Wessolek, G. (2012). Heavy metal pattern and soluteconcentration in soils along the oldest highway of theworld—the AVUS Autobahn. Environmental Monitoringand Assessment, 184, 6469–6481.

Kubrak, O. I., Lushchak, O. V., Lushchak, J. V., Torous, I. M.,Storey, J. M., Storey, K. B., & Lushchak, V. I. (2010).Chromium effects on free radical processes in goldfish tis-sues: comparison of Cr(III) and Cr(VI) exposures on oxida-tive stress markers, glutathione status and antioxidant en-zymes. Comparative Biochemistry and Physiology Part CToxicology and Pharmacology, 152, 360–370.

Laureysens, I., Blust, R., De Temmerman, L., Lemmens, C., &Ceulemans, R. (2004). Clonal variation in heavy metal accu-mulation and biomass production in a poplar coppice culture:I. Seasonal variation in leaf, wood and bark concentrations.Environmental Pollution, 131, 485–494.

Lettens, S., Vandecasteele, B., De Vos, B., Vansteenkiste, D., &Verschelde, P. (2011). Intra- and inter-annual variation of Cd,Zn, Mn and Cu in foliage of poplars on contaminated soil.Science of Total Environment, 409, 2306–2316.

Lushchak, O. V., Kubrak, O. I., Nykorak, M. Z., Storey, K.B., & Lushchak, V. I. (2008). The effect of potassiumdichromate on free radical processes in goldfish: pos-sible protective role of glutathione. Aquatic Toxicology,87, 108–114.

Madejón, P., Marañón, T., Murillo, J. M., & Robinson, B. (2004).White poplar (Populus alba) as a biomonitor of trace ele-ments in contaminated riparian forests. EnvironmentalPollution, 132, 145–155.

Madejón, P., Ciadamidaro, L.,Marañón, T., &Murillo, J.M. (2013).Long-term biomonitoring of soil contamination using poplartrees: accumulation of trace elements in leaves and fruits.International Journal of Phytoremediation, 15, 602–614.

Malayeri, B. E., Noori, M., & Jafari, M. (2012). Using the pollenviability and morphology for fluoride pollution biomonitor-ing. Biological Trace Element Research, 147, 315–319.

Nazzal, Y., Rosen, M. A., & Al-Rawabdeh, A. M. (2013).Assessment of metal pollution in urban road dusts from select-ed highways of the Greater Toronto Area in Canada.Environmental Monitoring and Assessment, 185, 1847–1858.

Paal, J., Degtjarenko, P., Suija, A., & Liira, J. (2013). Vegetationresponses to long-term alkaline cement dust pollution inPinussylvestris-dominated boreal forests—niche breadth along thesoil pH gradient. Applied Vegetation Science, 16, 248–259.

Pourrut, B., Shahid, M., Dumat, C., Winterton, P. & Pinelli, E.(2011). Lead uptake, toxicity, and detoxification in plants. InD. M. Whitacre (Ed.), Reviews of EnvironmentalContamination and Toxicology, 213 (pp. 113–136),Springer Science+Business Media.

Princewill-Ogbonna, I. L., & Ogbonna, P. C. (2011). Heavy metalcontent in soil and medicinal plants in high traffic urban area.Pakistan Journal of Nutrition, 10(7), 618–624.

Prus-Głowacki, W., Chudzińska, E., Wojnicka-Półtorak, A.,Kozacki, L., & Fagiewicz, K. (2006). Effects of heavy metalpollution on genetic variation and cytological disturbances inthe Pinus sylvestris L. population. Journal of AppliedGenetics, 47, 99–108.

Rotreklová, O. (2008). Hieracium subgen. Pilosella: pollen stain-ability in sexual, apomictic and sterile plants. Biologia, 63,61–66.

Sun, S.-Q., He, M., Cao, T., Yusuyin, Y., Han, W., & Li, J.-L.(2010). Antioxidative responses related to H2O2 depletion inHypnum plumaeforme under the combined stress induced byPb and Ni. Environmental Monitoring and Assessment, 163,303–312.

Swaileh, K. M., Matani, M., & Hussein, R. M. (2006). Heavymetals in urban roadside plants from Amman, Jordan.Bulletin of Environmental Contamination and Toxicology,77, 445–450.

Sytar, O., Kumar, A., Latowski, D., Kuczynska, P., Strzałka, K., &Prasad, M. N. V. (2013). Heavy metal-induced oxidativedamage, defense reactions, and detoxification mechanismsin plants. Acta Physiologiae Plantarum, 35, 985–999.

Trivedi, A. K., & Ahmad, I. (2011). Effects of chrysotile asbestoscontaminated soil on crop plants. Soil and SedimentContamination: An International Journal, 20, 767–776.

Van Nevel, L., Mertens, J., Staelens, J., De Schrijver, A., Tack, F.M. G., De Neve, S., et al. (2011). Elevated Cd and Zn uptakeby aspen limits the phytostabilization potential compared tofive other tree species. Ecological Engineering, 37, 1072–1080.

Vollenweider, P., Bernasconi, P., Gautschi, H.-P., Menard, T., Frey,B., & Günthardt-Goerg, M. S. (2011). Compartmentation ofmetals in foliage of Populus tremula grown on soils withmixed contamination. II. Zinc binding inside leaf cell organ-elles. Environmental Pollution, 159, 337–347.

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