Introduction 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2004 Vol. 47, Št. 2: 75-81 
Sprejeto (accepted): 2004-09-17 
Pollen grain bioassay for environmental contamination 
biomonitoring 
Biomonitoring polucije okolja z analizo poškodovanosti pelod-
nih zrn 
Jasna PARADIŽ and Milan LOVKA 
National Institute of Biology Ljubljana, Večna pot 111, SI-1000 Ljubljana, 
Slovenija; tel: +386(0)14233388, e-mail: jasna.paradiz@guest.arnes.si 
Abstract. Pollen grain bioassay near some highways was performed in 45 
plant species for identification of environmental genotoxic impact on naturally 
growing plants due to environmental traffic contaminants. A relationship between 
the duration of heavy traffic on particular road sections and the degree of develop-
mental and morphological changes of pollen grains was indicated. Increased fre-
quency of pollen deformation after more than 20 years of traffic influence was 
established, indicating the applicability of pollen grain bioassay for genotoxic risk 
assessment of chronic low leve! contamination impact in natura! habitats. 
Key Words: naturally growing bioindicator plants, traffic contaminants, pollen 
grain deformation 
Izvleček. V biomonitoringu onesnaženosti okolja zaradi prometa smo anal-
izirali stopnjo poškodovanosti pelodnih zrn pri 45 vrstah rastlin z naravnih rastišč 
ob naših avtocestah. Ugotovili smo, da je stopnja poškodovanosti peloda na 
poskusnih lokalitetah povezana s trajanjem prometa na posameznih avtocestnih 
odsekih, povečan obseg poškodovanosti peloda je bil pri biondikatorskih rastlinah 
na rastiščih, obremenjenih z vplivi prometa več kot 20 let. Ti rezultati kažejo, da je 
analiza poškodovanosti pelodnih zrn uporabna za sledenje kroničnih vplivov nizk-
ih nivojev zračne polucije z genotoksičnimi agensi in oceno ogroženosti rastlin na 
naravnih rastiščih. 
Ključne besede: samonikle bioindikatorske rastline, onesnaževanje z 
izpušnimi plini, poškodovanost pelodnih zrn. 
Environmental contamination caused by highway traffic has been identified as a serious ecolog-
ical problem. Air contaminants produced by motor traffic and released along the highways and main 
76 Acta Biologica Slovenica, 47 (2), 2004 
road connections are particularly hazardous to puhlic health and the environment. Low concentra-
tions of many environmental pollutants are considered as harmless, but for the genotoxic contami-
nants there is no safe <lose or concentration, and environmental hazard identification is required. 
Naturally growing plants are exposed to a changing mixture of environmental contaminants, and 
when used as bioindicators of long lasting exposure to complex pollution impact they have a key role 
in genotoxic risk evaluation of low contamination levels in natura! habitats. 
Our previous observation in plant natura! habitats with elevated contamination load (PARADIŽ & 
LOVKA 1998, 1999a, 1999b, 2000) and literature <lata (KORMUTAK 1996, MIČIETA & MURIN 1996, 
MULCAHY 1981, MURIN 1995) suggest that with many different species, plant bioassay results could 
give a more complex information about the pollution threats in the environment. Using 45 species of 
naturally growing plants in pollen grain bioassay near some highways in Slovenia, we have per-
formed screening of genotoxicity for risk identification of environmental impact of heavy traffic in 
natura! habitats. 
Material and Methods 
Experimental sites near some highways in Slovenia, conceming the duration of traffic on the spe-
cific road sections were chosen for biomonitoring with pollen grain bioassay. At 8 localities 
(Bizovik, Arja vas, Vransko, Čebulovica, Divača, Dolenja vas, Zajčica, and Sela) plants in their habi-
tats had been exposed to traffic influence for the period up to 10 years, at 2 localities (Povodje and 
Brdo) for 10 to 20 years, and at 5 localities (Gabrje, Ivanje selo, Ravnik, Trebnje and Žiče) for 20 to 
30 years. Localities at Komen, Predmeja, Pugled, and Škofja Loka with no obvious traffic pollution 
sources were used for the comparison. 
For pollen grain bioassay flower buds were sampled in 45 species of plants growing naturally at 
experimental and control sites (Tab. 1) on 18th, 241h, and 31 st of March, 7th of April, 19th and 26th of 
May, 2nd, 9th, 17th and 23rd of June 1999. The samples were fixed in ethanol acetic acid mixture (3:1), 
then kept at -200C in a refrigerator. 
Microscopic slides of fully developed anthers were made by staining in aceto--carmine. 
Generally, five slides from five different flowers per bioindicator species at the site were examined. 
In at least 300 pollen grains per slide, stained (viable) and empty (sterile) grains, as well as pollen 
size and shape deformations were observed under an optical microscope. Deformation frequencies 
of pollen grains, as well as portions of morphological changes in relation to the duration of highway 
traffic for up to 10 years, for 10-20 years and for 20-30 years are presented. 
Results 
In naturally growing bioindicator plants at the control sites, over 95% of stained (fertile) pollen 
grains were observed. In plants at experimental sites near highways, deformation frequency of pollen 
grains increased with the duration of traffic influence. The frequency of empty (sterile) grains 
increased up to 10% after 30 years of traffic influence (Fig. 1). Smaller size (for about 1/3 of the nor-
mal one) as well as shape deformations were less frequent than empty not stained grains, however, 
their portion increased with the duration of highway traffic as well (Tab. 2). 
J. Paradiž and M. Lovka: Pollen grain bioassay for environmental... 77 
Table 1. List of bioindicator species for pollen grain bioassay within sampling periods from March to June 
at experimental sites near highways and the control sites. Duration of traffic influence for particular road 
sections in brackets is stated. 
Tabela l. Seznam rastlinskih vrst vključenih v analizo poškodovanosti pelodnih zrn od marca do junija na 
poskusnih lokalitetah ob odsekih avtocest in kontrolnih lokalitetah. V oklepajih je navedeno trajanje 
prometa na posameznih odsekih avtocest. 
Sampling Bioindicator plants 
March Anemone nemorosa L. 
Capsella bursa-pastoris (L.) Med. 
CornusmasL. 
Corydalis cava (L.) Schweigger & Koerte 
Corylus avellana L. 
Crocus albiflorus Kit. ex Schult. 
Crocus napolitanus Mord. & Loisel. 
Crocus variegatus Hoppe & Hornsch. 
Daphne mezereum L. 
Erythronium dens-canis L. 
Galanthus nivalis L. 
Hacquetia ep ipactis (Scop.) DC. 
Helleborus niger L. 
Helleborus odorus W. & K. ex Willd. 
Lamium purpureum L. 
Leucojum vernum L. 
Omphalodes verna Moench 
Oxalis acetosella L. 
Polygala chamaebuxus L. 
Potentilla erecta (L.) Riiuschel 
Pulmonaria officinalis L. 
Symphytum officinale L. 
Tussilago farfara L. 
Vinca minor L. 
April/May Campanu/a rapunculus L. 
Crocus napolitanus Mord. & Loisel. 
Erythronium dens-canis L. 
Lamium orvala L. 
Lamium purpureum L. 
Melittis melissophyllum L. 
Myosotis sylvatica Ehrh. ex Hoffm. 
Oxalis acetosella L. 
Polygala chamaebuxus L. 
Potemilla erecta (L.) Riiuschel 
Ranunculus acris L. 
Ranunculus repens L. 
Symphytum officinale L. 
Trifolium pratense L. 
Trifolium repens L. 
Vinca minor L. 
June Calamintha grandiflora (L.) Moench 
Campanula rapunculus L. 
Coronilla emeroides Boiss. & Sprun. 
Coronilla varia L. 
Dianthus san uineus Vis. 
Erigeron annuus (L.) Pers. 
Experimental and control sites 
Bizovik(!), Arja vas (2), Trebnje (30) 
Arja vas (2) 
Komen, Žiče (23) 
Komen 
Povodje ( 14) 
Dolenja vas (4), Gabrje (25) 
Bizovik(!), Povodje (14), Brdo (18), Trebnje (30) 
Komen 
Trebnje (30) 
Brdo (18) 
Komen 
Ravnik (27) 
Pugled 
Pugled, Dolenja vas (4) 
Arja vas (2) 
Povodje (14) 
Ravnik (27) 
Arja vas (2) 
Pugled 
Čebulovica (4) 
Povodje (14), Ziče (23), Ivanje selo (27) 
Žiče (23) 
Vransko (2), Divača (4) 
Vransko (2) 
Sela (7) 
Trebnje (30) 
Sela (7), Povodje (14), Brdo (18) 
Povodje (14) 
Bizovik (1), Povodje (14) 
Pugled 
Povodje (14), Brdo (18) 
Trebnje (30) 
Pugled 
Pugled 
Bizovik (1) 
Sela (7), Povodje (14) 
Bizovik (1) 
Sela (7) 
Bizovik (1) 
Sela (7) 
Ravnik (27) 
Skofja Loka 
Vransko (2) 
Komen 
Komen 
kofja Loka 
78 
Coronilla emeroides Boiss. & Sprun. 
Coronilla varia L. 
Dianthus sanguineus Vis. 
Erigeron annuus (L.) Pers. 
Filipendula vulgaris Moench 
Geranium sylvaticum L. 
Lilium martagon L. 
Lysimachia nummularia L. 
Muscari racemosum (L.)Mill. em. Lam. & DC. 
Potentilla erecta (L.) Rauschel 
Ranunculus acris L. 
Ranunculus bulbosus L. 
Ranunculus repens L. 
Sanicula europaea L. 
Symphytum officinale L. 
Valeriana officinalis L. 
12 
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Acta Biologica Slovenica, 47 (2), 2004 
Vransko (2) 
Komen 
Komen 
SkofjaLoka 
Komen 
Ravnik (27) 
Gabrje (25) 
Škofja Loka 
Komen 
Škofja Loka 
Predmeja, Škofja Loka, Arja vas (2) 
Dolenja vas (4), Zajčica (4) 
Gabrje (25) 
Predmeja 
Arja vas (2) 
Divača (4) 
O Control 
[J 1 0Years 
• 20 Years 
III 30 Years 
March April/May 
Sampling period 
June 
Figure l. Pollen grain bioassay in naturally growing plants at experimental sites near highways and the con-
trol sites. 
Slika 1. Poškodovanost peodnih zrn pri naravno rastočih rastlinah na poskusnih lokalitetah ob avtocestah in 
kontrolnih lokalitetah. 
J. Paradiž and M. Lovka: Pollen grain bioassay for environmental ... 79 
Table 2. Portion (%) of size (small) and shape deformations of pollen grains in naturally growing plants near 
highways and the control. 
Tabela 2. Delež (%) malih in deformiranih pelodnih zrn pri naravno rastočih rastlinah ob avtocestah in kon-
troli. 
Traffic March April/May June 
influence Size Shape Nr. Size Shaee Nr. Size Shaee Nr. 
Control 7,2 0,1 7 4,2 0,0 3 3,4 0,1 11 
10 Years 5,6 4,1 10 5,0 3,2 9 13,3 1,8 5 
20 Years 9,5 2,0 5 4,5 11,7 5 
30 Years 8,7 11,8 9 1,5 1,7 2 4,6 6,3 4 
Nr. - Number of bioindicator species used in the pollen grain bioassay 
Discussion 
The genotoxic risk of environmental contarnination due to increasing traffic density and enlarged 
highway network in our country is of concem to both puhlic health and the environment. P!ants in 
their habitats are exposed to a broad spectrum of ecological and pollution risks, and they as bioindi-
cators of long lasting exposure to genotoxic effects in the environment are used in biomonitoring pol-
lution in view of environmental risk assessment (BESSONOVA & al. 1996, GICHNER & VELEMINSKY 
1999, JABEEN & ABRAHAM 1996, KORDYUM & SID0RENK0 1997, M0NARCA & AL. 1999, MULLER & 
AL. 1991, PARADIŽ & LOVKA 1998, 1999a, 1999b, 2000). 
In genotoxic risk identification, reduced pollen viability is one of the serious pollution impacts 
on reproductive capacity of plants that might link to ecological consequences. When the amount of 
stained pollen grains decrease Jess than 10%, pollen fertility of warm season species is not serious-
ly affected (TEDESCO & AL. 2002), but under normal climatic conditions pollen abortion in plants is 
Jess than 5% (MURIN 1995). This is in agreement with our results in bioindicator plants; the average 
frequency of 3% of empty (sterile) pollen grains at the control sites was established. 
At experimental sites near highways, the frequency of empty pollen grains (generally due to 
irregularities during meiosis) in bioindicator plants increased with the duration of traffic to 10% after 
30 years of traffic influence (Fig. 1), as well as the portion of size and shape deformations (Tab. 2). 
These results indicate that the genotoxic impact of long lasting environmental pollution might be 
related with traffic contaminants. 
When naturally growing plants for biomonitoring genotoxicity of pollutants are used, synergy 
with other environmental factors, such as meteorological conditions could not be excluded. With suc-
cessive sampling times at experimental sites near highways, the influence of extreme temperature or 
drought in sumrner on the results (Fig. 1) could be verified. But generally, prolonged sampling peri-
od for pollen grain bioassay is needed because of blooming season of various plant bioindicators. 
On the basis of the present results, pollen grain bioassay for continuous biomonitoring could be 
suggested, in addition to screening of detailed cytogenetical damage in plants, as well as the routine 
measurements of atmospheric contaminants for risk assessment of traffic pollution impact in the 
environment. 
80 Acta Biologica Slovenica, 47 (!), 2004 
Acknowledgements 
This work was part of a project supported by the Ministry of Science and Technology, Slovenia. 
Povzetek 
Ugotavljali smo vpliv škodljivih onesnaževalcev prometa na stopnjo poškodovanosti pelodnih 
zrn pri naravno rastočih rastlinah ob avtocestah. V raziskavo smo vključili 45 rastlinskih vrst in 
jemali cvetne popke od marca do julija za opazovanje sprememb v razvoju in obliki pelodnih zrn. 
Vzorčili smo na lokalitetah: Bizovik, Arja vas, Vransko, Čebulovica, Divača, Dolenja vas, Zajčica in 
Sela z vplivi prometa do 10 let; Povodje in Brdo z vplivi prometa 10 do 20 let; Gabrje, Ivanje selo, 
Ravnik, Žiče in Trebnje z vplivi prometa 20 do 30 let. Za primerjavo rezultatov smo uporabili 
lokalitete, oddaljene od glavnih prometnih cest (Komen, Predmeja, Pugled in Škofja Loka). 
Na poskusnih lokalitetah ob posameznih odsekih avtocest, kjer se odvija promet več kot 20 let smo 
ugotovili povečano stopnjo poškodovanosti pelodnih zrn pri naravno rastočih rastlinah. To kaže na 
uporabnost analize poškodovanosti pelodnih zrn za ugotavljanje dolgotrajnega vpliva zračne polucije 
na rastline v naravnem okolju. Z nadaljevanjem tovrstnih analiz bi doprinesli rezultate o škodljivih 
vplivih nizkih nivojev kontaminacije za oceno stopnje ogroženosti rastlin obremenjenih rastišč. 
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