VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

Analele Universităţii Ovidius 

Seria: BIOLOGIE – ECOLOGIE 

Volumul 14, anul 2010 

VOLUM OMAGIAL 

Ovidius University Annals 

BIOLOGY – ECOLOGY Series 

Volume 14, year 2010 

OVIDIUS UNIVERSITY PRESS

Analele Universităţii Ovidius, Seria Biologie – Ecologie 

Volumul 14 (2010) 

VOLUM OMAGIAL 

(dedicat împlinirii a 20 de ani de la înfiinţarea Facultăţii 

de Ştiinţe ale Naturii şi Ştiinţe Agricole) 

Redactor Şef 

Prof. univ. dr. Marian Traian GOMOIU 

Membru corespondent al Academiei Române 

mtg@datanet.ro 

Redactori 

Conf. univ. dr. Marius FĂGĂRAŞ Prof. univ. dr. Rodica BERCU 

fagaras_marius@yahoo.com rodicabercu@yahoo.com 

Mail address: Faculty of Natural and Agricultural Sciences, “Ovidius” University of Constanţa, Aleea 

Universităţii nr. 1, corp B, Constanţa RO-900470, România, Tel. 0241605060/ Fax: 0241606432, 

contact@stiintele-naturii.ro. 

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© 2010 Ovidius University Press ISSN 1453–1267

Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010) 

Contents 

Limitative mycotic factors for some plants from the Bulgarian coast of the Black Sea 

Gavril NEGREAN………………………………………………………………………................................ 

The medicinal plants of Provadiisko Plateau 

Dimcho ZAHARIEV, Desislav DIMITROV………………………………………………………………... 

The plants with protection statute, endemites and relicts of the Shumensko Plateau 

Dimcho ZAHARIEV, Elka RADOSLAVOVA……………………………………………………………... 

A characteristic of model habitats in south Dobrudja 

Dimcho ZAHARIEV………………………………………………………………………..……………….. 

Floristic aspects of the Hills of Camena village (Tulcea county) 

Marius FĂGĂRAŞ............................................................................................................................................ 

Identification of some rose genitors with resistance to the pathogens agents attack 

Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU, Elena DOROFTEI ………... 

Preliminary data on Meledic-Mânzăleşti Natural Reserve (Buzău county, Romania) 

Daciana SAVA, Mariana ARCUŞ, Elena DOROFTEI……………………………………………………… 

Contributions to the biometrical and phytobiological study on wild garlic 

Mariana LUPOAE, Dragomir COPREAN, Rodica DINICĂ, Paul LUPOAE………………………………. 

Dinitrophenyl derivates action on wheat germination 

Cristina Amalia DUMITRAŞ -HUŢANU ………………………….……………………………………….. 

The action of some insecticides upon physiological indices in Rana (Pelophylax) ridibunda 

Alina PĂUNESCU, Cristina M. PONEPAL, Octavian DRĂGHICI, Alexandru G. MARINESCU.............. 

Changes of some physiological parameters in Prussian carp under the action of some fungicide 

Maria C. PONEPAL, Alina PĂUNESCU, Alexandru G. MARINESCU, Octavian DRĂGHICI................... 

Cytogenetic effects induced by manganese and lead microelements on germination at Triticum 

aestivum L. 

Elena DOROFTEI, Maria Mihaela ANTOFIE, Daciana SAVA, Marioara TRANDAFIRESCU................... 

Problems of the harmonizing environmental legislation at the compartment “Pisces” in 

the Republic of Moldova 

Petru COCIRTA, Olesea GLIGA ………………………………………………………………………….... 

Biodiversity conservation in Constanţa county 

Silvia TURCU, Marcela POPOVICI, Loreley JIANU..................................................................................... 

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Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010) 

The present situation of the nose horned viper populations (Vipera ammodytes montandoni 

Boulenger 1904) from Dobrudja (Romania and Bulgaria) 

Marian TUDOR……………………………………………………………………………………………… 

Body size variation in Rana temporaria populations inhabiting extreme environments 

Rodica PLĂIAŞU, Raluca BĂNCILĂ, Dan COGĂLNICEANU…………………………………………… 

Utilization of epifluorescence microscopy and digital image analysis to study some morphological 

and functional aspects of prokariotes 

Simona GHIŢĂ, Iris SARCHIZIAN, Ioan ARDELEAN……………………………………………………. 

Changes in bacterial abundance and biomass in sandy sediment microcosm supplemented with 

gasoline 

Dan Răzvan POPOVICIU, Ioan ARDELEAN................................................................................................. 

The formation of bacterial biofilms on the hydrophile surface of glass in laboratory static 

conditions: the effect of temperature and salinity 

Aurelia Manuela MOLDOVEANU, Ioan I. ARDELEAN............................................................................... 

The clinical utility of additional methods in effusions evaluation 

Ana Maria CREŢU, Mariana AŞCHIE, Diana BADIU, Natalia ROŞOIU………………………………….. 

Spatio-temporal dynamics of phytoplankton composition and abundance from the Romanian Black 

Sea coast 

Laura BOICENCO…………………………………………………………………………………………… 

Aspects regarding the biodiversity of the aquatic and semi-aquatic heteroptera in the lakes situated 

in the middle basin of the Olt River 

Daniela Minodora ILIE..................................................................................................................................... 

Program of prevention and control of fungus infestation of grain and fodder, human and animal 

protection against mycotoxins 

Ioan Aurel POP, Augustin CURTICĂPEAN, Alin GULEA, Cornel PODAR, Iustina LOBONTIU.............. 

Data on the dynamics of some microbial groups in soils with different trophic status in Cumpăna 

region (Dobrudja) 

Elena DELCĂ………………………………………………………………………………………………... 

The agricultural potential of phosphogypsum waste piles 

Lucian MATEI……………………………………………………………………………………………….. 

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Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010 

LIMITATIVE MYCOTIC FACTORS FOR SOME PLANTS FROM THE 

BULGARIAN COAST OF THE BLACK SEA 

Gavril NEGREAN 

Universitatea din Bucureşti, Grădina Botanică, Şoseaua Cotroceni nr. 32, Bucureşti 

__________________________________________________________________________________________ 

Abstract: We present a list of 119 parasitic fungi collected in Bulgaria from the following groups: 

Peronosporales, Ascomycetes, Uredinales, Ustilaginales, Agaricales, Polyporales, Gasteromycetales and Fungi 

Anamorphici. An alien rust new for Bulgaria is also found (Puccinia komarovii). 

There are also some commentaries regarding the rare plants guested by different fungi; other fungi may 

contribute to the diminishing of the damages produces by some weeds; we draw attention about some foreign 

fungi with invasive character. 

Keywords: New parasitic fungi for Bulgaria, invasive fungi, matrix nova, Dobrogea, Bulgaria. 

__________________________________________________________________________________________ 

1. Introduction. 

Following our preoccupa-tion regarding the 

limitative factors for the vascular plants on the 

Black Sea, we present the results of our 

investigations on the Bulgarian Dobrogean Black 

Sea side. Our observations from the previous years 

were published within several notes [1, 2, 3, 4, 19]. 

2. Material and Methods. 

The fungi were collected from the areas nearby 

the sea side between Duranculac and the 

embouchure of the Batovo valley, in April and June 

2006 and April – October 2008. A very small 

amount of fungi collectetd from other areas of 

Bulgaria, they also listed. The big majority are 

coming from the Dobrich district. The materials 

were collected on the way and their conditioning 

was donje in conformity with the usual techniques 

and determinated by help of the instruments we had 

at our disposal [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. 

The nomenclature of the authors of the hosts 

after Flora Romaniae [15] and Flora Europaea [16, 

17]. The conditionated and determined materials 

were deposited in the Herbarium of the University 

from Bucureşti [BUC] and partially in the 

Herbarium of the Botanic Institute from Sofija 

[SOM]. The list is alphabetically coordinated, on 

big groups offungi and the coronims from North to 

South. 

3. Results and Discussions 

In these two years mentioned, there were 

collected 217 specimens, representing the analized 

groups of fungi (Table 1). Apparently, a number of 

16 combinations fungus – host plant („matrix 

nova”), incase of the Peronosporales (Table 2), and 

were not indicated since Bulgaria [14]. Among 

Erysiphaceae, 19 combinations [8], alike species 

have not been found in Bulgaria. Likewise, a 

number of 15 combinations between Uredinales [7] 

do not seem to be cited from Bulgaria. Puccinia 

komarovii rust, guesting the alien plant Impatiens 

parviflora DC. is new for the Bulgarian mycobiota. 

Sozological aspects. 

Following a long cohabitation (co evolution) 

between fungi and their hosts there has been created 

an equilibrum, so that we have barely noticed 

ruptures of this equilibrum. Among the rare guested 

plants, we mention the following: Astragalus 

cornutus (important damages locally), Buglossoides 

arvensis subsp. sibthorpiana, Centaurea 

salonitana, Centaurea thracica, Clypeo-la 

jonthlaspi, Dianthus leptopetalus, Euphorbia 

myrsinites, Gypsophila pallasii, Hieracium bauhinii, 

Leymus racemosus subsp. sabulosus, Limonium 

ISSN-1453-1267 © 2010 Ovidius University Press

Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) 

meyeri, Onosma rigidum, Potentilla taurica, Rhagadiolus 

stellatus, Scilla bithynica, Sherardia arvensis 

L. subsp. maritima etc. 

Some plants are extremely important from the 

sozological point of view, taking into consideration 

that they are in contact with new climatic 

conditions being subjected to the eventual 

speciation phenomena. It will be the case of Astragalus 

cornutus, Beta trigyna, Carduus pycnocephalus, 

Euphorbia dobrogensis, Medicago arabica, 

Pimpinella peregrina, Plumbago europaea, Ranunculus 

oxyspermus, Rumex tuberosus subsp. tuberosus, 

Scilla bithynica etc. 

Some fungi can have a certain play in 

diminishing the population of some weeds, 

contributiong this way to the diminution of the 

damages they produce, such as Amaranthus retroflexus, 

Avena fatua, Bassia scoparia, Carduus 

acanthoides, Lycium barbarum, Malva sylvestris, 

Picris echioides (carantin plant), Rumex patientia 

etc. 

I collecting some alien fungi with invasiv 

character in present (Puccinia malvacearum, Erysiphe 

mougeotii, Puccinia helianthi) or in future 

(Puccinia komarovii, Puccinia pelargonii-zonalis). 

The hyperparasite Ampelomyces quisqua-lis, 

also contributes the diminution of the damnages 

produced by some mildew, there have been registered 

some cases. 

Last but not least, we consider that the fungi 

have their right to live. 

4. Conclusions. 

From the 115 fungi collec-ted from the seaside 

of the Bulgarian Dobrogea, the most majority are 

plants parasites. Most of them belong the groups: 

Peronsporales, Erysiphaceae, Uredinales and Fungi 

Anamorphici. The results are important ones: a new 

adventitious parasite rust for the Bulgarian 

mycobiota and a number of 50 combinations from 

Bulgaria apparently not odentiofied in their form by 

now. We ascertained that following a long coexistence, 

between fungi and their hosts, the plants, 

there has been created a rather stable equilibrium. 

4 

LIST OF SPECIES 

PERONOSPORALES 

Albugo amaranthi (Schwein.) O. Kuntze 

(Wilsonia bliti (Biv.) Thines), matrix: 

Amaranthus retroflexus L. - Camen Briag, 

centrum, in locis ruderalis, 43º27′20.83″N, 

28º33′04.63″E, alt. circa 35m, 23 X 2008, G. 

Negrean (11.594) [BUC]. Cavarna S, prope hotel, 

in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, 

alt. circa 30 m, 10 VIII 2008, G. Negrean (11.484) 

[BUC]. 

Albugo candida (Pers.) Roussel, matrix: 

Alyssum desertorum Stapf - Bălgarevo E, Cap 

Caliacra W 2 km, in herbosis et petrosis, 14 IV 

2006, G. Negrean (7065c) [BUC]. 

Alyssum hirsutum Bieb. - Duranculac E, ad littore 

Mare Nigrum, in locis ruderalis, 43º41′908″N, 

28º34′300″E, alt. circa 5 m, 11 IV 2008, G. 

Negrean (10.220) [BUC]. 

Camelina rumelica Velen. - Cavarna E, in 

herbosis, 12 IV 2008, G. Negrean (10.256) [BUC]. 

Capsella bursa-pastoris (L.) Medicus - Balcic, 

centrum, in cortis moscheii, ruderal, 13 IV 2006, G. 

Negrean (7043) [BUC]. Balcic, centrum, ruderal, 4 

VI 2006, G. Negrean (7252) [BUC]. Sofija S, prope 

Hotel Vitosha (N), ruderal, 24 VI 2006, G. Negrean 

(7370). 

Clypeola jonthlaspi L. - Bălgarevo E, ut Cap 

Caliacra, in herbosis, 14 IV 2006, G. Negrean 

(7072a) [BUC]. Cavarna E, in herbosis, 

43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12 

IV 2008, G. Negrean (10.291) [BUC]. 

Sisymbrium loeselii L. - Sofija S, prope Univ. 

Technica, 21 VI 2006, G. Negrean (7323) [BUC]. 

Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 

2006, G. Negrean (7382) [BUC]. 

Sisymbrium orientale L. s. l. - Duranculac E, ad 

littore Mare Nigrum, in locis ruderalis, 

43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V 

2008, G. Negrean (10.436) [BUC]. 

Albugo portulacae (DC. ex Duby) O. Kuntze, 

matrix: 

Portulaca oleracea L. subsp. oleracea - Sozopol, 

in arenosis, 42º24′05.22″N, 27º42′33.32″E, alt. 

circa 15 m, 9 VIII 2008, G. Negrean (11.888) 

[BUC].

Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010) 

Albugo tragopogonis (DC.) Gray, matrix: 

Xeranthemum annuum L. - Cavarna E, in 

herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa 

60 m, 12 IV 2008, G. Negrean (11.504) [BUC]. 

Ecrene N, ad oram rivuli Batova, in arenosis, 

43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 13 IV 

2006, G. Negrean (7050) [BUC]. 

Bremia lactucae Regel, matrix: 

Carduus acanthoides L. - Bălgarevo E, Cap 

Caliacra, vers E, ut Mare Nigrum, in herbosis 

ruderalis, 15 IV 2006, G. Negrean (7074a) [BUC]. 

Cavarna SW, Bojorets S, Caliacria, in locis 

ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. circa 

46 m, 22 X 2008, G. Negrean (11.521) [BUC]. 

Picris echioides L. - Camen Briag, in locis 



Rhagadiolus stellatus (L.) Gaertn. - Rusalca, sub 

platous prope littore Mare Nigrum, in abruptis et 

silvis, 43º24′733″N, 28º29′780″E, alt. circa 60 m, 

10 V 2008, G. Negrean (10.459) [BUC]. 

Crepis pulchra L. - Bălgarevo E, Cap Caliacra N, 

sinistra vallis Bolata-Dere, in herbosis, 

43º22′59.37″N, 28º28′18.08″E, alt. circa 2 m, 4 VI 

2006, G. Negrean (7397) [BUC]. 

Hyaloperonospora parasitica (Pers.: Fr.) 

Constant., matrix: 

Alyssum desertorum Stapf - Bălgarevo E, Cap 

Caliacra W 2 km, in herbosis et petrosis, 14 IV 

2006, G. Negrean (7065b) [BUC]. 

Hyaloperonospora tribulina (Pass.) Constant. 

(Peronospora tribulina Pass.), matrix: 

Tribulus terrestris L. - Cavarna SW, Caliacria, in 

locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, alt. 

circa 46 m, 22 X 2008, G. Negrean (11.511) 

[BUC]. 

Peronospora aestivalis H. Syd., matrix: 

Melilotus sp. - Balcic W, in herbosis, 3 VI 2006, 

G. Negrean (7225) [BUC]. 

Peronospora alsinearum Casp., matrix: 

Stellaria media (L.) Vill. s. l., Ecrene N, ad oram 

rivuli Batova, 43º21′10.43″N, 28º04′31.74″E, alt. 

circa 2 m, 13 IV 2006, G. Negrean (7820) [BUC]. 

Peronospora alta Fuckel, matrix: 

Plantago major L. subsp. major, Sofija S, prope 

Hotel Moskva, 22 VI 2006, G. Negrean (7328) 

[BUC]. 

Peronospora arborescens (Berk.) de Bary, 

matrix: 

5 

Papaver dubium L. - Bălgarevo E, Cap Caliacra, 

vers E, ut Mare Nigrum, in herbosis ruderalis, 


IV 2006, G. Negrean (7079) [BUC]. 

Peronospora astragalina Syd., matrix: 

Astragalus hamosus L. - Crapetz E, prope Cap 

Crapetz, solo arenoso, 43º38′23.64″N, 

28º34′25.86″E, alt. circa 6 m, 12 IV 2008, G. 


Peronospora calotheca de Bary, matrix: 

Galium aparine L. - Crapetz E, prope Cap Crapetz, 

solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. circa 


Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo 

terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa 15 

m, 12 IV 2008, G. Negrean (10.270) [BUC]. 

Peronospora conglomerata Fuckel, matrix: 

Erodium ciconium (L.) L’Herit. - Cavarna S, in 

arenosis, sub Collina Cheracman, 12 IV 2008, G. 

Negrean (10.241) [BUC]. Cavarna SW, Bojorets S, 

Caliacria, in locis ruderalis, 43º25′03.47″N, 

28º16′48.25″E, alt. circa 46 m, 22 X 2008, G. 


Peronospora farinosa (Fr.) Fr., matrix: 

Bassia scoparia (L.) A. J. Scott, Sofija S, cartier 

S, ruderal, 22 VI 2006, G. Negrean (7330) [BUC]. 

Chenopodium album (Boiss.) Kuntze - Camen 

Briag, Motel, in locis ruderalis, 43º27′13.83″N, 

28º33′04.96″E, alt. circa 25 m, 7 VI 2008, G. 


Chenopodium opulifolium Schrad. ex Koch & Ziz 

- Cavarna S, prope hotel, in locis ruderalis, 


VIII 2008, G. Negrean (11.484) [BUC]. 

Peronospora ficariae L.R. Tul. ex de Bary, 

matrix: 

Ranunculus ficaria L. subsp. calthifolius 

(Reichenb.) Arcangeli - Balcic W, in locis umbrosis, 

15 IV 2006, G. Negrean (7090) [BUC]. 

Peronospora medicaginis-minimae 

Gaponenko, matrix: 

Medicago lupulina L., Sofija S, prope Hotel 

Vitosha, ruderal, 22 VI 2006, G. Negrean (7321) 

[BUC]. 

Peronospora sherardiae Fuckel, matrix: 

Sherardia arvensis L. subsp. maritima (Griseb.) 

Soják - Bălgarevo E, Cap Caliacra, in herbosis, 

43º22′03.97″N, 28º26′57.08″E, alt. circa 25 m, 12


IV 2008, G. Negrean (10.234) [BUC]. Balcic W, in 

herbosis, 15 IV 2006, G. Negrean (7087a) [BUC]. 

Peronospora tribulina Pass. = Hyaloperonospora 

tribulina (Pass.) Constant. 

Peronospora valerianellae Fuckel, matrix: 

Valerianella sp. - Crapetz E, prope Cap Crapetz, 

solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. 

circa 6 m, 12 IV 2008, G. Negrean (10.245) [BUC]. 

Peronospora viciae (Berk.) de Bary, matrix: 

Vicia sativa L. subsp. nigra (L.) Ehrh. - 

Duranculac S, in herbosis, 43º39′53.06″N, 

28º31′15.26″E, alt. c. 12 m, 13 IV 2006, G. 

Negrean (7036) [BUC]. Rusalca NNE, in herbosis, 


2008, G. Negrean (10.277) [BUC]. 

Plasmopara nivea (Unger) J. Schröt. 

(Plasmopara umbelliferarum (Casp.) J. Schröt. ex 

Wartenw.), matrix: 

Aegopodium podagraria L., Sofija S, Montes 

Vitosha, in herbosis subalpinis, 23 VI 2006, G. 

Negrean (7353) [BUC]. 

ASCOMYCOTA 

Blumeria graminis (DC. ) Speer, matrix: 

Avena fatua L. - Rusalca, sub platou prope littore 

Mare Nigrum, in herbosis, 43º25′116″N, 

28º31′126″E, alt. circa 10 m, 7 VI 2008, G. 


Aegilops lorentii Hochst. - Rusalca, sub platou 

prope littore Mare Nigrum, in herbosis, 


2008, G. Negrean (10.465) [BUC]. 

Hordeum bulbosum L. Dobrogea, Shabla E, ad 

littore Mare Nigrum, in locis herbosis, 


2008, G. Negrean (11.572) [BUC]. 

Daldinia concentrica (Bolton) Ces. & De Not. 

- matrix: in lignos, Duranculac E, ad littore Mare 

Nigrum, in locis arenosis, 43º41′908″N, 

28º34′300″E, alt. circa 5 m, 11 IV 2008, Leg. P. 

Anastasiu, det. G. Negrean (11.593) [BUC]. 

Epichloe typhina (Pers.: Fr.) Tul., matrix: 

Dactylis glomerata L. s. l., Sofija S, prope Hotel 

Moskva, in herbosis, 22 VI 2006, G. Negrean 

(7331). 

Erysiphe alphitoides (Griffon & Maubl.) U. 

Braun & S. Takam. (Microsphaera alphitoides 

Griffon & Maubl.), matrix: 

6 

Quercus pubescens Willd. - Bălgarevo E, 

Rusalca, prope littore Mare Nigrum, sub abruptum, 

10 VIII 2008, G. Negrean (11.503) [BUC]. 

Quercus robur L., Sofija S, prope Hotel Vitosha 

(N), ruderal, 24 VI 2006, G. Negrean (7379). 

Erysiphe aquilegiae DC., matrix: 

Aquilegia vulgaris L., - Camen Briag, centrum, in 

locis ruderalis, subspont., 43º27′20.83″N, 


Negrean (11.536, T) [BUC; CL]. 

Erysiphe artemisiae Grev., matrix: 

Artemisia vulgaris L. - Camen Briag, centrum, in 


circa 35 m, 23 X 2008, G. Negrean (11.532) [BUC]. 

Erysiphe astragali DC., matrix: 

Astragalus hamosus L. - Rusalca, prope littore 




Erysiphe buhrii U. Braun, matrix: 

Gypsophila pallasii Ikonn. - Bălgarevo E, dextra 

vallis Bolata Dere, prope littore Mare Nigrum, in 

herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35 

m, 20 VII 2006, G. Negrean (11.450) [BUC]. 

Erysipe cichoracearum DC., matrix: 

Centaurea salonitana Vis. - Rusalca N, ad littore 



Negrean (10.720) [BUC]. Yailata, platou prope 

littore Mare Nigrum, in saxosis, 43º26′552″N, 

28º32′930″E, alt. circa 25 m, 8 VIII 2008, G. 

Negrean (11.472) [BUC]. Bălgarevo E, Cap 

Caliacra, in herbosis, 43º22′982″N, 28º26′4599″E, 

alt. circa 72 m, 8 VI 2008, G. Negrean (10.753) 

[BUC]. Balcic E, supra Tuzlata, in herbosis 

abruptis, 8 VI 2008, G. Negrean (10.747) [BUC]. 

Crepis foetida L. subsp. rhoeadifolia (Bieb.) 

Čelak., Sofija S, prope Hotel Vitosha (N), ruderal, 

24 VI 2006, G. Negrean (7375). 

Crepis pulchra L., Sofija S, prope Hotel Vitosha 

(N), ruderal, 24 VI 2006, G. Negrean (7371). 

Lactuca viminea L. - Rusalca, prope littore Mare 

Nigrum, in locis herbosis et petrosis, 43º24′733″N, 

28º29′776″E, alt. circa 45 m, 10 V 2008, G. 


Tragopogon dubius Scop., Sofija S, prope Hotel 

Vitosha (N), ruderal, 24 VI 2006, G. Negrean 

(7376).


Erysiphe cruciferarum Opiz ex L. Junell, 

matrix: 

Alliaria petiolata (Bieb.) Cavara & Grande, 

Sofija S, prope Hotel Moskva, ruderal, 22 VI 2006, 

G. Negrean (7324). 





Brassica nigra C. Koch - Bălgarevo SE, ad 

littore Mare Nigrum, sub abruptum, prope ferma 

pisciculturae, in locis herbosis, 43º25′02.83″N, 

28º31′00.18″E, alt. circa 5 m, 10 VIII 2008, G. 


Erysiphe cynoglossi (Wallr.) U. Braun, matrix: 

Buglossoides arvensis (L.) I. M. Johnston subsp. 

sibthorpiana (Griseb.) R. Fernandes - Balcic W, 

Cap Caliacra, in herbosis, 3 VI 2006, G. Negrean 

(7295) [BUC]. 

Echium italicum L. subsp. pyramidatum (DC.) . - 

Bălgarevo SE, Cap Caliacra, in herbosis, 



Echium vulgare L., Sofija S, prope Hotel Vitosha 

(N), ruderal, 25 VI 2006, G. Negrean (7383) 

[BUC]. 

Onosma rigidum Ledeb. - Yailata, prope littore 

Mare Nigrum, in abruptis, 43º26′552″N, 


Negrean (11.124, A) [BUC]. 

Erysiphe depressa (Wallr.) Schltdl – A, 

matrix: 

Onopordum acanthium L. - Cavarna SW, Bojorets 

S, Caliacria, 43º25′03.47″N, 28º16′48.25″E, alt. 

circa 46 m, 22 X 2008, G. Negrean (11.525) [BUC]. 

Erysiphe galeopsidis DC. = Neoerysiphe 

galeopsidis (DC.) U. Braun 

Erysipe heraclei DC., matrix: 

Myrrhoides nodosa (L.) Cannon - Rusalca, sub 

platou prope littore Mare Nigrum, in herbosis, 


2008, G. Negrean (10.465) [BUC]. 

Scandix pecten-veneris L. subsp. pecten-veneris - 

Rusalca, sub platou prope littore Mare Nigrum, in 


m, 7 VI 2008, G. Negrean (10.650) [BUC]. 

Tordylium maximum L. - Rusalca, sub platou 

prope littore Mare Nigrum, in fossa viam, 

7 

43º25′120″N, 28º31′125″E, alt. circa 12 m, 12 IV 

2008, G. Negrean (11.413) [BUC]. 

Torilis nodosa (L.) Gaertner - Rusalca, ad littore 

Mare Nigrum, in herbosis prope marem, 

43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI 

2008, G. Negrean (10.784) [BUC]. 

Erysiphe knautiae Duby, matrix: 

Knautia arvensis (L.) Coulter, Sofija S, prope 

Hotel Vitosha, in herbosis, 22 VI 2006, G. Negrean 

(7335) [BUC]. 

Erysiphe lycopsidis R. Y. Zheng & G. Q. 

Chen, matrix: 

Anchusa arvensis (L.) Bieb. - Shabla E, ad littore 

Mare Nigrum, in locis herbosis, 43º24′954N″, 



Erysiphe mougeotii (Lév.) de Bary, matrix: 

Lycium barbarum L. - Cavarna, centrum, 


X 2008, G. Negrean (11.543) [BUC]. 

Erysiphe polyphaga Hammarl. = Golovinomyces 

orontii (Castagne) V. P. Heliuta 

Erysiphe ranunculi Grev., matrix: 

Ranunculus constantinopolitanus (DC.) D’Urv. - 

Ecrene N, ad oram rivuli Batova, 43º21′10.43″N, 

28º04′31.74″E, alt. circa 2 m, 3 VI 2006, G. 


Erysiphe thesii L. Junell, matrix: 

Thesium alpinum L., Sofija S, Montes Vitosha, in 

herbosis subalpinis, 23 VI 2006, G. Negrean (7347) 

[BUC]. 

Erysiphe trifolii Grev., matrix: 

Medicago arabica L. - Camen Brjag, centrum, in 


circa 35 m, 23 X 2008, G. Negrean (11.531, A) 

[BUC]. 

Melilotus officinalis (L.) Pallas - Cavarna SW, 

Bojorets S, Caliacria, in locis ruderalis, 


X 2008, G. Negrean (11.522) [BUC]. Sofija S, 

prope Hotel Moskva, in herbosis, 22 VI 2006, G. 


Trifolium hybridum L. subsp. elegans (Savi) 

Aschers. & Graebn., Sofija S, Hortus Botanicus, in 

herbosis, 20 VI 2006, G. Negrean (7313) [BUC]. 

Golovinomyces orontii (Castagne) V. P. 

Heliuta (Erysiphe polyphaga Hammarl.), matrix:


Sedum sarmentosum Bunge - Camen Brjag, motel, 

cult., 43º27′20.40″N, 28º33′13.07″E, alt. circa 35 


Neoerysiphe galeopsidis (DC.) U. Braun 

(Erysiphe galeopsidis DC.), matrix: 

Lamium amplexicaule L. Duranculac E, ad littore 


28º34′300E, alt. circa 5 m, 9 V 2008, G. Negrean 

(10.434) [BUC]. Cavarna S, prope hotel, in locis 



Balcic, centrum, ruderal, 4 VI 2006, G. Negrean 

(7252) [BUC]. Sofija, centrum, 20 VI 2005, G. 


Phyllactinia guttata (Wallr.: Fr.) Lév., matrix: 

Fraxinus angustifolia Vahl subsp. oxycarpa 

(Bieb. ex Willd.) Franco & Rocha Afonso - 

Cavarna, centrum, 43º26′13.44″N, 28º20′36.38″E, 

alt. circa 127 m, 23 X 2008, G. Negrean (11.549) 

[BUC]. 

Podosphaera euphorbiae (Castagne) U. Braun 

& S. Takam., matrix: 

Euphorbia esula L. subsp. orientalis (Boiss.) 

Molero & Rovira, 1992 (Euphorbia esula subsp. 

tommasini-ana (Bertol.) Nyman) - Bălgarevo SE, 

Cap Caliacra, in herbosis, 43º23′02.13″N, 



Sawadea bicornis (Wallr.: Fr.) Homma 

(Uncinula bicornis (Wallr.: Fr.) Lév., matrix: 

Acer negundo L., subspont., Sofija S, prope Univ. 

Technica, 21 VI 2006, G. Negrean (7319) [BUC]. 

Sphaerotheca aphanis (Wallr.) U. Braun, 

matrix: 

Geum urbanum L., Sofija S, prope Hotel 

Moskva, 22 VI 2006, G. Negrean (7324) [BUC]. 

Sphaerotheca fugax Penz. & Sacc., matrix: 

Erodium ciconium (L.) L’Hér. - Bălgarevo E, Cap 

Caliacra, in herebosis, 3 VI 2006, G. Negrean 

(7256) [BUC]. 

Geranium rotundifolium L. - Bălgarevo E, Cap 

Caliacra, vers E, ut Mare Nigrum, in herbosis 

ruderalis, 15 IV 2006, G. Negrean (7071a) [BUC]. 

Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo 

terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa 


Taphrina deformans (Berk.) Tul., matrix: 

8 

Prunus dulcis Miller - Balcic, prope Hortus 

Botanicus, cult., 2 V 2008, G. Negrean (10.343) 

[BUC]. 

Prunus persica (L.) Batsch - Bălgarevo E, Cap 

Caliacra, cult., 30 IV 2008, G. Negrean (10.358) 

[BUC]. 

Taphrina pruni Tul., matrix: 

Prunus cerasifera Ehrh. - Shabla E, prope littore 

Mare Nigrum, 43º33′755″N, 28º35′250″E, alt. circa 

3 m, 9 V 2008, G. Negrean (11.112) [BUC]. 

Prunus domestica L. - Balcic, prope hotel 

Eisberg, cult., 30 IV 2008, G. Negrean (10.359) 

[BUC]. 

Venturia geranii (Fr.) G. Winter, matrix: 

Erodium ciconium (L.) L’Herit. - Duranculac E, 

ad littore Mare Nigrum, in locis ruderalis, 

43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV 

2008, G. Negrean (10.224) [BUC], 9 V 2008, G. 


UREDINALES: 

Aecidium euphorbiae Link, O, I, matrix: 

Euphorbia agraria Bieb. - Bălgarevo E, ut Cap 

Caliacra, in herbosis, 14 IV 2006, G. Negrean 

(7064a) [BUC]. Bălgarevo E, Cap Caliacra, in 


25 m, 12 IV 2008, G. Negrean (10.239 [BUC]. 

Euphorbia myrsinites L. - Bălgarevo E, vallis 

Bolata Dere, terra rossa, 43º23′08.40″N, 


Negrean (10.268) [BUC]. Bălgarevo E, Cap 


alt. circa 72 m, 12 IV 2008, G. Negrean (10.236) 

[BUC]. 

Euphorbia nicaeensis All. s. l. - Crapetz, ut 

faleza, in herbosis, 12 IV 2008, G. Negrean 

(10.240) [BUC]. 

Euphorbia seguieriana Necker - Duranculac E, ad 

littore Mare Nigrum, in locis arenosis, 

43º41′908″N, 28 º34′300″E, alt. circa 5 m, 11 IV 

2008, G. Negrean (10.217) [BUC]. 

Melampsora euphorbiae (Ficinus & C. 

Schub.) Castagne, matrix: 

Euphorbia helioscopia L. - Duranculac E, ad 



2008, G. Negrean (10.431, ii, iii) [BUC]. 

Duranculac E, ad littore Mare Nigrum, in locis


ruderalis, 43º41′908″N, 28º34′300″E, alt. circa 5 m, 

6 VI 2008, G. Negrean (10.598) [BUC]. Rusalca, 

sub platou prope littore Mare Nigrum, in herbosis, 


2008, G. Negrean (10.661) [BUC]. Bălgarevo E, 

Cap Caliacra, in herbosis, 43º22′982″N, 


Negrean (10.233, ii, iii) [BUC]. Bălgarevo E, inter 

Cap Caliacra et Bolata Dere, prope littore Mare 

Nigrum, in herbosis, 43º22′405-23′144″N, 

28º27′928-965″E, alt. circa 30 m, 11 V 2008, G. 

Negrean (10.497) [BUC]. Cavarna, sub Montes 

Cheracman, 30 IV 2008, G. Negrean (10.376) 

[BUC]. 

Phragmidium mucronatum (Pers.) Schltdl, 

matrix: 

Rosa canina L. - Rusalca, prope littore Mare 

Nigrum, in herbosis et petrosis, 43º24′733″N, 


Negrean (10.462, i) [BUC]. 

Phragmidium potentillae (Pers.) P. Karst., 

matrix: 

Potentilla pedata Nestler - Bălgarevo E, Cap 

Caliacra, in herebosis, 4 VI 2006, G. Negrean 

(7294) [BUC]. 

Potentilla taurica Willd. - Shabla E, ad littore 

Mare Nigrum, in locis herbosis, 43º24′954″N, 



Phragmidium sanguisorbae (DC.) Schröt., 

matrix: 

Sanguisorba minor Scop. s. l., Sofija S, prope 

Hotel Vitosha (N), ad viam ferream, 26 VI 2006, G. 


Phragmidium violaceum (Schultz) G. Winter, 

matrix: 

Rubus candicans Weihe ex Rchb. - Bălgarevo E, 

Rusalca, in herbosis, supra Mare Nigrum, 


VII 2008, G. Negrean (11.406) [BUC]. 

Rubus discolor Weihe & Nees - Bălgarevo SE, 

prope littore Mare Nigrum, sub abruptum, prope 

ferma pisciculturae, in locis herbosis, 


VIII 2008, G. Negrean (11.498, ii) [BUC]. 

Puccinia allii (DC.) F. Rudolphi, matrix: 

Allium tauricum (Besser ex Rchb.) Grossh. – 

Bălgarevo E, Cap Caliacra, 43º22′982″N, 

9 


Negrean (10.288) [BUC, ii]. 

Allium sp. - Ecrene N, ad oram rivuli Batova, in 

herbosis, 43º20′55.87″N, 28º04′25.15″E, alt. circa 1 

m, 3 VI 2006, G. Negrean (7235) [BUC]. 

Puccinia asperulae-cynanchicae Wurth, 

matrix: 

Asperula tenella Heuffel ex Boiss. - NE Bulgaria: 

prov. Burgas: Aitos, in petrosis, 11 VI 1973, G. 

Negrean [BUC]. 

Puccinia calcitrapae DC., matrix: 

Carduus pycnocephalus L. - Rusalca, sub platou 

prope littore Mare Nigrum, in saxosis, 


2008, G. Negrean (10.698, iii) [BUC]. 

Centaurea thracica (Janka) Hayek - Bălgarevo E, 

dextra vallis Bolata Dere, in herbosis, 43º23′144″N, 

28º27′965E″, alt. circa 34 m, 6 VI 2008, G. 


Puccinia cesatii J. Schröt., matrix: 

Dichanthium ischemum (L.) Roberty - Bălgarevo 

SE, Cap Caliacra, 43º23′02.13″N, 28º26′53.26″E, 


[BUC]. 

Puccinia crepidis J. Schröt., matrix: 

Crepis foetida L. subsp. rhoeadifolia (Bieb.) 

Čelak. - Bălgarevo SE, Cap Caliacra, in herbosis 

ruderalis, prope Archer (Boris Caragea), 


2008, G. Negrean (10.231) [BUC]. Cap Caliacra, in 

herbosis, 43º22′03.97″N, 28º27′57.08″E , alt. circa 

30 m, 8 VI 2008, G. Negrean (10.753) [BUC]. 

Puccinia dobrogensis Săvul. & O. Săvul. (?= 

Puccinia caucasica Savelli), matrix: 

Iris pumila L. - Bălgarevo E, Cap Caliacra, in 


25 m, 8 VI 2008, G. Negrean (11.120) [BUC]. 

Puccinia gladioli (Requien) Cast., I, matrix: 

Valerianella costata (Steven) Betcke - Bălgarevo 

E, Cap Caliacra, situs archaeologicus, in herbosis, 

14 IV 2006, G. Negrean (7817) [BUC]. 

Valerianella sp. - Bălgarevo E, Cap Caliacra, in 


25 m, 12 IV 2008, G. Negrean (10.255, i) [BUC]. 

Cavarna E, in herbosis, 13 IV 2008, G. Negrean 

(10.261) [BUC]. 

Puccinia graminis DC., matrix: 

Festuca drymeja Mert. & Koch - distr. Shumen: 

Shumen W, Platous Shumen, in silvis,



VII 2008, G. Negrean (11.603). 

Puccinia helianthi Schwein., matrix: 

Helianthus annuus L. „Florae Pleno”, cult. - 

Camen Briag, centrum, 43º27′20.83″N, 



Puccinia hieracii Mart., matrix: 

Hieracium bauhinii Besser, Sofija S, prope Hotel 

Vitosha, 22 VI 2006, G. Negrean, matrix conf. 

Krahuleć (Pruhonice) (7322) [BUC]. 

Puccinia isiacae (Thüm.) G. Wint., matrix: 

Cardaria draba (L.) Desv. subsp. draba - 

Duranculac E, ad littore Mare Nigrum, in ruderatis, 

43º41′908″N, 28 º34′300″E, alt. circa 5 m, 9 V 

2008, G. Negrean (10.426, i) [BUC]. 

Puccinia komarovii Tranzschel, matrix: 

Impatiens parviflora DC. - Sofija S, Hotel 

Vitosha (N), Parc, subspont. ad viam ferream, 24 

VI 2006, G. Negrean (7380) [BUC; SOM]. Fungus 

adventivus novus Bulgariae. Originally from 

Central Asia, alien in Europe. In Romania on 

Impatiens parviflora DC., subspont. in Botanical 

Garden of Cluj-Napoca, Valea Pârîul Ţiganilor, 

46°51′46″N, 23°35′20″E, alt. 347 m, 5 VII 1993, G. 

Negrean [BUCM 129.306]. 

Puccinia lapsanae Fuckel, matrix: 

Lapsana communis L. - Sofija S, prope Hotel 

Vitosha (N), ad viam ferream, 24 VI 2006, G. 


Puccinia malvacearum Bertero ex Mont. – iii, 

matrix: 

Althaea hirsuta L. - Duranculac E, ad littore 


28º33′922″E, alt. circa 5 m, 6 VI 2008, G. Negrean 

(11.121) [BUC]. Bălgarevo E, Cap Caliacra, in 



Malva sylvestris L. - Shabla E, ad littore Mare 

Nigrum, in locis herbosis, 43º24′954″N, 

28º30′061″E, alt. circa 10 m, 9 V 2008, G. Negrean 

(10.444) [BUC]. Yailata, prope littore Mare 

Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. 

circa 25 m, 10 V 2008, G. Negrean (11.123) 

[BUC]. Rusalca NNE, in herbosis, 43º25′34.94″N, 


Negrean (10.276) [BUC], 43º24′733″N, 


Negrean (10.460) [BUC], Rusalca, ad littore Mare 

10 

Nigrum, in locis herbosis, 43º24′954″N, 


Negrean () [BUC]. Balcic, centrum, in cortis 

moscheii, ruderal, 13 IV 2006, G. Negrean (7044) 

[BUC]. Nesebăr, in arenosis ruderalis ad littore 

Mare Nigrum, 5 VI 2006, G. Negrean (7439) 

[BUC]. 

Puccinia minussensis Thüm., matrix: 

Lactuca tatarica (L.) C. A. Meyer - Duranculac 

E, ad littore Mare Nigrum, in ruderatis, 


2008, G. Negrean (10.429) [BUC], 6 VI 2008, G. 


Puccinia pachyphloea Syd. & H. Syd., matrix: 

Rumex tuberosus L. subsp. tuberosus - Bălgarevo 

E, Cap Caliacra N, Bolata-Dere, in herbosis, 4 VI 

2006, G. Negrean (7396) [BUC]. 

Puccinia pelargonii-zonalis Doidge, matrix: 

Pelargonium ×hortorum auct. - Sofija S, Hortus 

Botanicus, in caldaria, cult. 42º43′..N, 23º19′...E, 20 

VI 2006, G. Negrean (7311) [BUC; SOM]. 

Puccinia phragmitis (Schumach.) Körn., 

matrix: 

Rumex patientia L. s. l. - Duranculac E, ad littore 

Mare Nigrum, in ruderatis, 43º41′908″N, 



Puccinia pimpinellae (F. Strauss) Link, 

matrix: 

Pimpinella peregrina L. - Yailata, prope littore 

Mare Nigrum, in abruptis, 43º26′552″N, 


Negrean (10.465) [BUC]. Rusalca, sub platou prope 

littore Mare Nigrum, in herbosis, 43º25′116″N, 



Puccinia procera Dietel & Holw., matrix: 

Leymus racemosus (Lam.) Tzvelev subsp. 

sabulosus (Beb.) Tzvelev - Shabla E, ad littore 



(10.639, ii) [BUC]. 

Puccinia punctata Link, matrix: 

Galium verum L. subsp. verum - Bălgarevo SE, 

prope littore Mare Nigrum, sub abruptum, prope 

ferma pisciculturae, in locis herbosis, 



Puccinia recondita Dietel & Holw., matrix:


Aegilops cylindrica Host - Duranculac E, ad littore 



(10.601) [BUC]. Balcic W, Cap Caliacra, in 


Sofija S, prope Hotel Vitosha (N), ruderal, 24 VI 

2006, G. Negrean (7377). 

Aegilops geniculata Roth - Rusalca, platou prope 

littore Mare Nigrum, in locis herbosis et petrosis, 


2008, G. Negrean (10.696, iii) [BUC]. Bălgarevo E, 

Cap Caliacra, in herbosis, 43º22′405″N, 


Negrean (10.765, iii) [BUC]. 

Anchusa sp. - Bălgarevo E, Cap Caliacra, in 


m, 12 IV 2008, G. Negrean (10.234) [BUC]. 

Bromus sterilis L. subsp. elegans - Sofija S, 

prope Hotel Vitosha, 22 VI 2006, G. Negrean 

(7317) [BUC]. 


Bălgarevo SE, Cap Caliacra, 43º23′02.13″N, 



Clematis vitalba L.- Yailata, prope littore Mare 

Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt. 

circa 25 m, 10 V 2008, G. Negrean (10.777, i) 

[BUC]. Bălgarevo E, inter Cap Caliacra et Bolata 

Dere, prope littore Mare Nigrum, in herbosis, 

43º22′405-23′144″N, 28º27′928-965″E, alt. circa 50 

m, 11 V 2008, G. Negrean (10.506) [BUC]. Ecrene 

N, ad oram rivuli Batova, in arenosis, 

43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 3 VI 

2006, G. Negrean (7238) [BUC]. 

Puccinia sii-falcariae J. Schröt., matrix: 

Falcaria vulgaris Bernh. - Duranculac E, ad 



2008, G. Negrean (10.597) [BUC]. Shabla E, ad 

littore Mare Nigrum, in locis herbosis, 


2008, G. Negrean (10.441) [BUC]. Cavarna E, in 


Bălgarevo E, inter Cap Caliacra et Bolata Dere, 

prope littore Mare Nigrum, in herbosis, 43º22′405- 

23′144″N, 28º27′928-965″E, alt. circa 30 m, 11 V 

2008, G. Negrean (10.497) [BUC]. Bălgarevo E, 

Cap Caliacra N, Bolata-Dere, in herbosis, 6 VI 

2006, G. Negrean (7396) [BUC]. 

11 

Puccinia tanaceti DC., matrix: 

Artemisia absinthium L. - Duranculac E, ad littore 



(10.594) [BUC]. Cavarna SW, Caliacria, in locis 



Tranzschelia pruni-spinosae (Pers.) Dietel – 

(ii), iii, matrix: 

Prunus cerasifera Ehrh. - Duranculac NE, ad 

confines Bulgariae, 43º44′10.05″N, 28º33′24.68″E, 


[BUC; SOM]. 

Uromyces dianthi (Pers.: Pers.) Niessl, matrix: 

Dianthus leptopetalus Willd. - Bălgarevo E, 

sinistra vallis Bolata Dere, prope littore Mare 

Nigrum, in herbosis, 43º23′140″N, 28º28′000″E, 

alt. circa 35 m, 20 VII 2008, G. Negrean (11.476) 

[BUC]. 

Petrorhagia prolifera (L.) P. W. Ball & 

Heywood - Bălgarevo E, Cap Caliacra, in herbosis, 

4 VI 2006, G. Negrean (7256) [BUC]. 

Uromyces limonii (DC.) Lév., matrix: 

Limonium latifolium (Sm.) Kuntze - Yailata, ad 

littore Mare Nigrum, 43º26′131″N, 28º32′665″E, 


[BUC]. 

Limonium meyeri (Boiss.) Kuntze - Rusalca, sub 

platou, prope littore Mare Nigrum, in saxosis, 


2008, G. Negrean (10.690) [BUC]. Rusalca, sub 

platou, prope littore Mare Nigrum, in saxosis, 


VII 2008, G. Negrean (11.434) [BUC]. 

Uromyces lineolatus (Desm.) Schroet., matrix: 

Scirpus maritimus L. subsp. maritimus - Shabla 

NE, ad littore Mare Nigrum, in arenosis, 

43º24′954″N, 28º30′061″E, alt. circa 4 m, 20 VII 

2008, G. Negrean (11.578) [BUC]. 

Uromyce punctatus J. Schröt., matrix: 

Astragalus cornutus Pallas - Bălgarevo E, dextra 

vallis Bolata Dere, prope littore Mare Nigrum, in 


m, 20 VII 2008, G. Negrean (11.449) [BUC]. 

Uromyces rumicis (Schumach.) G. Winter, 

matrix: 

Rumex patientia L. s. l. - Camen Briag, centrum, 

in locis ruderalis, 43º27′20.83″N, 28º33′04.63″E,



[BUC]. 

Uromyces scutellatus (Pers.: Pers.) Lév., 

matrix: 

Euphorbia agraria Bieb. - Crapetz, ut faleza, in 


Euphorbia dobrogensis Prodan - Duranculac E, 

ad littore Mare Nigrum, in herbosis, 43º41′908″N, 

28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean 

(11.113) [BUC]. 

Euphorbia nicaeensis All. s.l. - Duranculac E, ad 

littore Mare Nigrum, in herbosis et petrosis, 


2008, G. Negrean (10.599) [BUC]. 

Euphorbia seguieriana Necker - Sofija S, prope 

Hotel Vitosha, 22 VI 2006, G. Negrean (7320). 

Uromyces trifolii-repentis Liro, matrix: 

Trifolium hybridum L. subsp. elegans (Savi) 

Aschers. & Graebn., Sofija S, Hortus Botanicus, in 


USTOMYCETES 

Entyloma calendulae (Oudem.) de Bary, 

matrix: 

Calendula officinalis L., Sofija S, cult., 22 VI 

2006, G. Negrean (7320). 

Microbotryum violaceoverrucosum 

(Brandenb. & Schwinn) Vánky, matrix: 

Silene bupleuroides Sm. s. l. - Yailata, platou 


43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII 

2008, G. Negrean (11.470) [BUC]. 

Microbotryum violaceum (Pers.: Pers.) G. 

Deml & Oberw. s. l., matrix: 

Silene latifolia Poiret subsp. alba (Miller) Greuter 

& Burdet - Shabla E, ad littore Mare Nigrum, in 

locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa 

10 m, 9 V 2008, G. Negrean (11.116) [BUC; CL]. 

Camen Brjag, centrum, in cortis, 43º27′20.83″N, 


Negrean (11.538) [BUC; CL]. 

Sorosporium saponariae F. Rudolphi, matrix: 

Silene bupleuroides Sm. s. l. - Yailata, platou 


43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII 

2008, G. Negrean (11.471) [BUC]. 

Ustilago cynodontis (Pass.) P. Henn., matrix: 

12 

Cynodon dactylon (L.) Pers. - Shabla NE, ad littore 

Mare Nigrum, in arenosis, 43º24′954N, 

28º30′061″E, alt. circa 4 m, 20 VII 2008, G. 

Negrean (11.584) [BUC]. Rusalca, sub platou prope 

littore Mare Nigrum, in Paliuretum, 43º25′116″N, 

28º31′126″E, alt. circa 10 m, 19 VII 2008, G. 

Negrean (11.417) [BUC]. Bălgarevo E, dextra 

vallis Bolata Dere, in herbosis, 43º23′144″N, 



Ustilago ornithogali (J. C. Schmidt & Kunze) 

J. G. Kühn, matrix: 

Gagea pusilla (F. W. Schmidt) Schult. & Schult. 

fil. - Cavarna E, in herbosis, 43º24′25.14″N, 



Ustilago vaillantii L.-R. Tul. & C. Tul., matrix: 

Scilla bithynica Boiss. - Ecrene N, ad oram rivuli 

Batova, in Alnetum, Fraxinetum pallisiae et 

Salicetum, in locis humidis, 43º20′55.58″N, 


Negrean (7056) [BUC; CL]. 

AGARICALES, POLYPORALES, GASTEROMYCETALES 

Dendrothele acerina (Pers.: Fr.) P. A. Lemke, 

matrix: 

Acer campestre L. - Rusalca, sub platou, prope 

littore Mare Nigrum, in silvis, 43º25′140″N, 



Fomitopsis pinicola (Sw.) P. Karst., matrix: 

Picea abies (L.) Karsten subsp. abies - Montes 

Vitosha, 23 VI 2006, G. Negrean (7824) [BUC]. 

Hymenochaete rubiginosa (Dicks.) Lév., 

matrix: 

Quercus robur L. - Sofija, Hotel Vitosha N, Hotel 

Moskva W, park, 24 VI 2006, G. Negrean (7389c) 

[BUC]. 

Lepista panaeolus (Fr.) P. Karsten - ad solum, 

Dobrogea, Cavarna SW, Bojorets S, Caliac- 

ria, in locis ruderalis, 43º25′03.47″N, 28º16′48.25″E, 

alt. circa 46 m, 23 X 2008, G. Negrean (12.077). 

Polyporus melanopus (Pers.) Fr., matrix: in 

lignos, distr. Shumen: Shumen W, Platous Shumen, 

in Fagetum, 43º14′55.96″N, 26º53′45.52″E, 


[BUC].


Polyporus varius Pers.: Fr., matrix: ad ramulos 

decidous, distr. Shumen: Shumen, park, 

43º16′12.73″N, 26º560′30.79″E, alt. circa 205 m, 

18 VI 2008, G. Negrean (11.586) [BUC]. 

Suillus bellinii (Inzenga) Watling, ad solum, 

sub Pinus nigra Arnold, cult., Dobrogea, Cavarna 

SW, Bojorets S, Caliacria, in abruptis et petrosis, 


X 2008, G. Negrean (11.592) [BUC; CL]. 

Trametes hirsuta (Wulfen) Pilát, matrix: in 

lignos, distr. Shumen: Shumen W, Platous 

Shumen, in Fagetum, 43º14′55.96″N, 

26º53′45.52″E, alt. circa 474 m, 18 VII 2008, G. 


Tulostoma brumale Pers.: Pers., ad solum, 

Duranculac E, ad littore Mare Nigrum, in arenosis 

maritimis, 43º40′296″N, 28º33′918″E, alt. circa 5 

m, 9 V 2008, comm. P. Anastasiu, det. G. Negrean 

& P. Anastasiu (10.426) [BUC]. 

Tulostoma squamosum Pers. - Ecrene N, ad 

oram rivuli Batova, in arenosis, 43º21′10.43″N, 



Volvariella gloiocephala (DC.: Fr.) Boekhout 

& Enderle - ad solum, Dobrogea, Cavarna SW, 



X 2008, G. Negrean (12.075). 

FUNGI ANAMORPHICI 

Ampelomyces quisqualis Ces., 

socio cum: Erysiphe cruciferarum Opiz ex 

L. Junell - matrix: 





Socio cum: Erysiphe cynoglossi (Wallr.) U. Braun, 

matrix: 


Bălgarevo SE, Cap Caliacra, in herbosis, 



Cercospora plumbaginea Sacc. & D. Sacc., 

matrix: 

Plumbago europaea L. - Cavarna S, prope hotel, 

in locis ruderalis, 43º21′17.41″N, 28º21′17.41″E, 

13 


[BUC]. 

Cercospora taurica Tranzsch., matrix: 

Heliotropium europaeum L. - Camen Briag, 

centrum, in locis ruderalis, 43º27′20.83″N, 


Negrean (11.591) [BUC; CL ]. Cavarna SW, 



X 2008, G. Negrean (11.529) [BUC]. 

Napicladium celtidis Cavara, matrix: 

Celtis australis L. - Cavarna S, prope hotel, in 


circa 30 m, 10 VIII 2008, G. Negrean (11.489) 

[BUC]. 

Ovularia obliqua (Cooke) Oudem., matrix: 

Rumex patientia L. s. l. - Duranculac E, ad littore 

Mare Nigrum, locis ruderatis, 43º41′908″N, 



Ramularia arvensis Sacc., matrix: 

Potentilla recta L., Sofija S, prope Hotel Vitosha, 

20 VI 2006, G. Negrean (7309) [BUC]. 

Ramularia beticola Fautrey & Lambotte, 

matrix: 

Beta trigyna Waldst. & Kit. - Rusalca, sub platou 

prope littore Mare Nigrum, 43º24′750″N, 



Ramularia centaureae Lindr., matrix: 

Centaurea salonitana Vis. - Bălgarevo E, Cap 


alt. circa 72 m, 8 VI 2008, G. Negrean (10.754) 

[BUC]. 

Ramularia libanotidis Bubák, matrix: 

Seseli campestre Besser - Rusalca, sub platou, 

prope littore Mare Nigrum, 43º25′116″N, 



Ramularia ranunculi-oxyspermi Lobik, 

matrix: 

Ranunculus oxyspermus Bieb. - Cavarna E, in 



5. References 

[1] NEGREAN Gavril, CONSTANTINESCU 

Ovidiu & DENCHEV Cvetomir M. 2004.


Addition to the Peronosprales of Bulgaria. 

Mycologia Balcanica 1(1): 69-72. 

[2] NEGREAN Gavril & DENCHEV Cvetomir M. 

2000. New record of Bulgarian parasitic fungi. 

Flora Mediterranea (Palermo) 10: 101-108. 


2002. New record of fungi from bulgarian 

Dobrudzha. Pp. 21-24. In: N. RANDJELOVIĆ 

(ed.), Proceedings of Sixth Symposium on Flora 

of Southeastern Serbia and Adjacent Territories, 

July 4-7, 2000, Sokobanja, Yugoslavia. Vuk 

Karadžić, Niš. 


2004. Addition to the Erysiphales of Bulgaria. 

Mycologia Balcanica 1(1): 63-66. 

[5] BRAUN Uwe. 1987. A monograph of the 

Erysiphales (powdery mildews). Beih. Nova 

Hedw. Heft 89. Berlin, Stuttgart: J. Cramer, 700 

pp., 316 fig. 

[6] BRAUN Uwe. 1995. The powdery mildews 

(Erysiphales) of Europe. Jena: Gustav Fischer 

Verlag, i-iv, 1-337 pp., ill. 112, ISBN 3-334- 

60994-4 (HB). 

[7] DENCHEV Cvetomir M. 1995. Bulgarian 

Uredinales. Mycotaxon 55: 405-465. 

[8] FAKIROVA Violeta Ilieva ● ФАКИРОВА 

Виолета Илиева. 1991. Fungi Bulgaricae 1 

tomus ordo Erysiphales ● Гъбите в България 1 

том разред Еrysiphales, red. princip. prof. Dr. I. 

Kovachevsky; edit. Simeon Vanev, Edit. Acad. 

Bulgaricae, Sofija, 154 pp. 

[9] MAJEWSKI T. 1977. Grzyby (Mycota), T. IX, 

Podstawczaki (Basidiomycetes), Rdzawniko we 

(Uredinales) I, Flora Polska, Warsawa - Kraków: 

Panstwowe Wydawnictwo Naukowe. 396 pp. 

[10] MAJEWSKI T. 1979. Grzyby (Mycota), T. XI, 

Podstawczaki (Basidiomycetes), Rdzawnikowe 

(Uredinales) II, Flora Polska, Warsawa - 

Kraków: Panstwowe Wydawnictwo Naukowe. 

463 pp. + Erata + 2 Pl. 

[11] SĂVULESCU T. 1953. Monografia 

Uredinalelor din Republica Populară Română ● 

Monographia Uredinalium Reipublicae 

Popularis Romanicae, vol. 1-2. Bucureşti: Edit. 

Academiei Române, 1166 pp. (vol. 1: 1-332 + ixxiv 

+ liii Pl. + 21 Tab.; vol. 2: 333-1168. /B: 

339-343/. 

[12] SĂVULESCU T. 1957. Ustilaginalele din 

Republica Populară Romînă ● Ustilaginales 

14 

Reipublicae Popularis Romanicae, vol. 1-2. 

Bucureşti: Edit. Academiei Romîne, 1168 pp. 

/vol. I: 1-545 pp; vol. II: 546-1170 pp., index: 

1141-1168/. 

[13] SCHOLLER M. 1996. Die Erysiphales, 

Pucciniales und Ustilaginales der Vorpommerschen 

Boddenlandschaft - Ökologisch-floristiche, 

florengeschichtliche und morphologisch-taxonomische 

Untersuchungen. Regensb. Mykol. Schriften 

6: 1-325. 

[14] VANEV Simeon Georgiev, DIMITROVA 

Evtimia Georgieva & ILIEVA Elena Ivanova ● 

ВАНЕВ Симеон Георгиев, ДИМИТРОВА 

Евтимия Георгиева & ИЛИЕВА Елена 

Иванова. 1993. Fungi Bulgaricae 2 tomus ordo 

Peronosporales ● Гъбите в България 2 Tом 

разред Peronosporales. Red. principali Prof. Dr. 

Ivan KOVACHEVSKI, Editit tomum, Violeta 

FAKI-ROVA. Sofija: Edit. Academiae 

Scientiarum Bulgaricae, 195 pp. + Erata, 1 fig., 1 

tab., 57 pl. ISBN 954-430-227-1 (t. 2). 

[15] SĂVULESCU T. (ed.). 1952-1976. Flora 

României ● Flora Romaniae. Bucureşti: Edit. 

Academiei Române. Vol. 1-13. 

[16] TUTIN T. G., BURGES N. A., CHATER A. 

O., EDMONDSON J. R., HEYWOOD V. H., 

MOORE D. M., VALENTINE D. H., WALTERS 

S. M. & WEBB D. A. (eds, assist. by 

J. R. AKEROYD & M. E. NEWTON; 

appendices ed. by R. R. MILL). 1993. Flora 

Europaea. 2nd ed. Vol. 1. Psilotaceae to 

Platanaceae. Cambridge: Cambridge University 

Press xlvi, 581 pp., illus. ISBN 0-521-41007-X 

(HB). 

[17] TUTIN T. G., HEYWOOD V. H., BURGES 

N. A., MOORE D. M., VALENTINE D. H., 

WALTERS S. M. & WEBB D. A. (eds). 1964- 

1980. Flora Europaea. Vols. 1-5. Cambridge: 

Cambridge University Press. 

[18] HOLMGREN Patricia K. & HOLMGREN 

Noel H. (ed.). 1992. Plant specialists index: Index 

to specialists in the systematics of plants and 

fungi based on data from Index Herbariorum 

(Herbaria), edition 8. Königstein: Koeltz 

Scientific Books, 1-394. [Regnum Vegetabile 

120], ISBN 3-87429-331-9 (HB). 

[19] NEGREAN G. 1992. Violeta Ilieva Fakirova, 

Fungi Bulgaricae 1 tomus ordo Erysiphales, red.


princip. prof. Dr. I. Kovachevsky; edit. Simeon 

Vanev, Edit. Acad. Bulgaricae, Sofija, 1991, 154 

pp.etc. Stud. Cerc. Biol., Ser. Biol. Veg. 44(2): 

196-197. /recenzie critică/. 

15 

Aknowledgments 

We thanks Mrs. Professor Paulina Anastasiu 

for the help given in order to draw this material and 

to Dr. Krahulec (Pruhonice) for confirming the 

identification of Hieracium bauhinii.


THE MEDICINAL PLANTS OF PROVADIISKO PLATEAU 

Dimcho ZAHARIEV, Desislav DIMITROV 

University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences, 

115 Universitetska Str., 9712, Shumen, Bulgaria, 

dimtchoz@yahoo.com 

_________________________________________________________________________________________ 

Abstract: Considerable taxonomical diversity of the medicinal plants of Provadiisko Plateau is established: 376 

species of vascular plants from 261 genera and 86 families. Most families (77.91%) and genera (98.85%) are 

represented in small numbers – 1 to 4. The analysis of their life form indicates that the geophytes dominante, 

followed by the groups of the phanerophytes and the hemi cryptophytes. These biological types are represented 

mainly by perennial herbaceous plants (53.19%) and annual herbaceous plants (12.77%). The largest percentage 

species are of the circumboreal type (36.17%). Among the medicinal plants, there are 4 endemites and 29 relicts. 

39 species with protection statute are described. The anthropophytes among the medicinal plants are 236 species 

(62.77%). 

Keywords: Provadiisko Plateau, medicinal plants, analysis of medicinal plants, protected species. 

______________________________________________________________________________________ 

1. Introduction 

In physiographic terms the Provadiisko Plateau 

belongs to the Danube hilly plain area, i.e. the 

Ludogorsko-Provadiiska subarea [1]. The Northern 

plateau border is the Provadiiska River; in the East it 

reaches to the Devnya Valley; in the South, the 

Provadiisko Plateau is separated from Roiaksko 

Plateau by Glavnica River; and finally, west of the 

Provaddisko Plateau is the Shumensko Plateau. The 

average altitude is 250 m. above sea level. The 

highest point is Sakartepe in the western parts of the 

plateau with its height of 389 m. The plateau is 

located in the Transcontinental climate region, district 

Dobrudjansko Plateau [2]. Winds are coming mostly 

from the North and Northeast. The average annual 

temperature is around 12°С. The average monthly 

temperatures are always positive. The temperature in 

January is the lowest (1.2°С) and in July – the highest 

(22.6°С). The minimum temperature rarely fall to 

18°С, and the average maximum temperature reaches 

27°С. The maximum rainfalls are in May and June 

and the minimum – in March and September. The 

annual amount of rainfalls is around 530 mm. 

Average humidity is around 76-77%; lowest in the 

summer (70%) and highest in the winter (82%) [3]. 

The soils, according to the FAO classification, are 

two types. The first type is calcic chernozems located 

on the slopes and in the areas with low slope. The 

second type is calvaric fluvisols located in the 

Provadiiska Valley [4]. 

In terms of its flora, the plateau belongs to the 

region of Northeastern Bulgaria. The vegetation 

includes: forests of Carpinus betulus L. and Quercus 

cerris L., partly with Carpinus orientalis Mill.; mixed 

forests of Carpinus betulus L. and Quercus cerris L., 

partly with Quercus dalechampii Ten., Acer 

campestre L., etc.; mixed forests of Tilia tomentosa 

Moench., with Carpinus betulus L. or Quercus cerris 

L., partly also with Quercus dalechampii Ten., Acer 

campestre L., etc.; forest and shrubs of Carpineta 

orientalis; mixed forests of Quercus cerris L., 

Quercus pubescens Willd. and Cotinus coggygria 

Scop., partly with a secondary prevalence of Cotinus 

coggygria Scop.; mixed forests of Fraxinus ornus L. 

and Carpinus orientalis Mill., partly of secondary 

origin; shrubs with prevalence of Paliureta spinachristi, 

combined with xerothermal frass communities 

mostly replacing xerothermal forest communities of 

Quercus cerris L. and Quercus frainetto Ten.; shrub 

and grass steppe and xerothermal communities; 

xerothermal grass communities with a prevalence of 

Dichantieta ischaemi, Poaeta bulbosae, Poaeta 

concinnae, Chrysopogoneta grylli and Ephemereta; 


The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) 

mesoxerothermal grass vegetation with a prevalence 

of Poa bulbosa L., Loium perenne L., Cynodon 

dactylon (L.) Pers., partly also Dichantium 

ischaemum (L.) Roberty and rarely Chrysopogon 

gryllus (L.) Trin., mostly in the village com 

monlands; mesophytous grass communities 

(meadows), replacing forests of Ulmus minor Mill., 

Fraxinus oxicarpa Willd., Quercus robur L., 

Quercus pedunculiflora C. Koch.; farm areas, 

replacing forests of Fagus sylvatica ssp. moesiaca 

(K. Maly) Hyelmq.; farm areas, replacing forests of 

Quercus dalechampii Ten.; farm areas, replacing 

forests of Ulmus minor Mill., Fraxinus oxicarpa 

Willd., Quercus pedunculiflora C. Koch. [5]. 

The first studies of the flora of the plateau have 

been conducted in the 1990s by Vasil Kovachev 

around Madara, Kaspitchan and Provadia [6]. The 

results are found in the first volume on Bulgarian 

flora [7] and its supplement [8]. Hermengild Shkorpil 

also conducted botanical researchin the vicinity of 

Provadia in the early twentieth century [6]. 

So far, data on the medicinal plants in the area of 

Provadiisko Plateau have been published by authors 

for the the territory of Municipality Provadia [9] and 

by Zahariev and Uzunov for the protected area 

Madarski rock wreaths [10]. 

The Provadiisko Plateau is a part of the protected 

zone Provadiisko-Roiaksko Plateau by Natura 2000, 

according to Council Directive 92/43/EEC of the 

European Community to protect natural habitats and 

of wild fauna and flora [11]. 

2. Material and Methods 

The field studies were conducted on the route 

method in 2007-2009. The names of the taxons are 

taken from the Flora of PR Bulgaria, Vol. І – Х [12]. 

The update of the taxons is consistent with APG II 

[13]. The life forms are presented by Raunkier [14]. 

In their determination was used Flora of PR Bulgaria, 

Vol. І – Х [12]. The biological types are presented by 

Kozuharov [15]. The floristic elements and endemites 

are presented by Asiov et all. [16]. The relicts are 

presented by Gruev and Kuzmanov [17], Peev [18], 

Boža et all. [19], Peev et all. [20]. The protection 

status is presented using the following documents: 

Council Directive 92/43/EEC of the European 

18 

Community to protect natural habitats and of wild 

fauna and flora [11], Convention on 

International Trade in Endangered Species of Wild 

Fauna and Flora (CITES) [21], Red book of PR 

Bulgaria [22], IUCN Red List for Bulgaria [23], 

Biological Diversity Act [24], Order for special 

arrangements for the conservation and use of 

medicinal plants [25]. The anthropophytes are 

presented by Stefanov and Kitanov [26]. 


As a result of the research of the medicinal plants 

of the Provadiisko Plateau 376 species of vascular 

plants from 261 genera and 86 families have been 

indetified. They represent 9.83% from all species, 

29.36% from all genera and 50.89% from all plant 

families in Bulgaria. 

Most families (77.91%) and genera (98.85%) are 

represented in small numbers: 1 to 4. 

Almost all families (86.05%) are represented 

with 1-4 genera. Only 13.95% from the families 

included 5 or more genera (Table 1). Most genera are 

found in the families: Asteraceae (28), Lamiaceae 

(22), Fabaceae (21), Rosaceae (15), Apiaceae (14) 

and Brassicaceae (12). 

Table 1. Families with greatest number of genera 

Families Genera 

Asteraceae 28 

Lamiaceae 22 

Fabaceae 21 

Rosaceae 15 

Apiaceae 14 

Brassicaceae 12 

Scrophulariaceae 8 

Ranunculaceae 8 

Caryophyllaceae 8 

Boraginaceae 7 

Poaceae 5 

Solanaceae 5 

Most families – 77.91% have 1-4 species. Only 

22.09% of the families are represented by 5 or more 

species (Table 2). Most species belong to the 

following families: Asteraceae (42), Lamiaceae (41),

Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010) 

Fabaceae (28), Rosaceae (26), Brassicaceae (15), 

Apiaceae (15), Ranunculaceae (12) and 

Scrophulariaceae (11). 

Almost all genera (98.85%) are represented by 1- 

4 species. Most species – more than 5 have only 

1.15% of the genera (Table 3): Centaurea, Geranium 

and Thymus. 

Table 2. Families with greatest number of species 

Families Species 

Asteraceae 42 

Lamiaceae 41 

Fabaceae 28 

Rosaceae 26 

Brassicaceae 15 

Apiaceae 15 

Ranunculaceae 12 

Scrophulariaceae 11 

Boraginaceae 9 

Caryophyllaceae 9 

Orchidaceae 7 

Geraniaceae 6 

Polygonaceae 6 

Solanaceae 6 

Aspleniaceae 5 

Oleaceae 5 

Poaceae 5 

Rubiaceae 5 

Salicaceae 5 

Table 3. Genera with greatest number of species 

Families Genera Species 

Asteraceae Centaurea 5 

Geraniaceae Geranium 5 

Lamiaceae Thymus 5 

In the analysis of the life forms were obtained the 

following results (Table 4): The phanerophytes (Ph) 

are represented by 86 species. The megaphanerophytes 

are represented by 10 species, the most 

common of which are: Acer pseudoplatanus L., 

Fraxinus excelsior L., Gleditsia triacanthos L., Pinus 

sylvestris L., Quercus frainetto Ten., Quercus robur 

L. 

19 

The mezophanerophytes are 35 species, of which 

essential are: Acer campestre L., Acer 

pseudoplatanus L., Carpinus betulus L., Fagus 

sylvatica L., Fraxinus ornus L., Tilia tomentosa 

Moench, Ulmus minor Mill. 

The microphanerophytes are 27 species, the most 

common of which are: Acer tataricum L., Cornus 

mas L., Corylus avellana L., Cotinus coggygria 

Scop., Crataegus monogyna Jacq., Hedera helix L., 

Ligustrum vulgare L., Paliurus spina-christi Mill., 

Prunus spinosa L., Rosa canina L., Rubus caesius L., 

Sambucus nigra L. 

The nanophanerophytes are 11 species, which 

are essential: Clematis vitalba L., Genista tinctoria 

L., Teucrium chamaedrys L., Teucrium polium L. 

The succulents are represented by 3 species: 

Sedum acre L., Sedum album L. and Sedum 

maximum (L.) Suter. 

Table 4. Life forms 

Group Subgroup Species 

Megaphanerophytes 10 

Mezophanerophytes 35 

Phanerophytes Microphanerophytes 27 

(Ph) Nanophanerophytes 11 

Epiphytes – 

Succulents 3 

Hamephytes (Ch) 5 

Hemi cryptophytes (H) 65 

Therophyte – hemi cryptophytes 45 

(Th-H) 

Cryptophytes Geophytes 126 

(Cr) 

Helophytes 1 

Hydrophytes – 

Therophytes (Th) 48 

The group of hamephytes (Ch) includes 5 

species: Dictamnus albus L., Ruscus aculeatus L., 

Satureia montana L., Thymus jankae Čelak., Thymus 

zygioides Griseb. 

The hemi cryptophytes (H) are 65 species, of 

which most common are: Agrimonia eupatoria L., 

Carlina vulgaris L., Cichorium intybus L., 

Clinopodium vulgare L., Echium vulgare L., 

Eryngium campestre L., Lotus corniculatus L., 

Marrubium peregrinum L., Plantago lanceolata L.,


Plantago media L., Polygala major Jacq., 

Ranunculus ficaria L., Salvia nemorosa L., Silene 

vulgaris (Moench) Garcke, Taraxacum officinale 

Web., Trifolium pratense L., Trifolium repens L., 

Verbena officinalis L., Viola odorata L. 

The transition group therophytes – hemi 

cryptophytes (Th-H) comprises 45 species, of which 

essential are: Alliaria petiolata (Bieb.) Cavara et 

Grande, Arctium lappa L., Capsella bursa-pastoris 

Moench., Daucus carota L., Erodium cicutarium (L.) 

L̀ Her., Heracleum sibiricum L., Malva sylvestris L., 

Plantago major L., Stellaria media (L.) Vill., 

Tordylium maximum L., Verbascum densiflorum 

Bertol., Viola tricolor L. 

The group of cryptophytes (Cr) is the largest and 

includes 127 species. Their significant proportion can 

be explained by the dominance of forest habitats 

within the plateau. Geophytes dominate with total of 

126 species; the most widespread of them are: 

Achillea millefolium L., Anemone ranunculoides L., 

Artemisia absinthium L., Artemisia vulgaris L., 

Chelidonium majus L., Convolvulus arvensis L., 

Coronilla varia L., Fragaria vesca L., Galanthus 

elwesii Hook. fil., Galanthus nivalis L., Geum 

urbanum L., Isopyrum thalictroides L., Potentilla 

argentea L., Sanguisorba minor Scop., Scilla bifolia 

L., Urtica dioica L. The helophytes is represented by 

one species only: Typha latifolia L. 

The therophytes (Th) are 48 species. The most 

widespread are: Galium aparine L., Lactuca serriola 

L., Lamium purpureum L., Lolium temulentum L., 

Melilotus officinalis (L.) Pall., Papaver rhoeas L., 

Parietaria lusitanica L., Xeranthemum annuum L. 

The largest group species in terms of biological 

types (Figure 1) are perennial plants (p) – 200 species 

(53.19%). Their dominance can be explained with the 

wide variety of communities and habitats within the 

plateau. 

The annual plants (a) are 48 species (12.77%), 

which can be explained by the presence of dry rocky 

terrain and arable lands on the plateau. 

The tree species (t) are 39 (10.37%). The next 

group includes shrubs (sh) – 29 species (7.71%). The 

transition group from annual to biennial plants (a-b) 

includes 19 species (5.05%). The biennial plants (b) 

are 13 species (3.46%). There are species from 

transition group from tree to shrubs (sh-t) with 12 

20 

species (3.19%) and species from transition group 

from biennial to perennial plants (b-p) – 11 species 

(2.92%). The largest group includes annual as well as 

perennial plants (a-p) and is represented by 5 species 

(1.33%). 

29 

12 

39 

200 

Fig. 1. Biological types 

The specific physiographic conditions on the 

Provadiisko Plateau determined considerable 

diversity of floristic elements. 7 different types of 

floristic elements are established (Table 5). The 

dominant elements are elements from circumboreal 

type – 136 species (36.17%), followed by European 

elements – 101 species (26.86%) and Mediterranean 

elements – 67 species (17.82%). The endemic 

component is represented by 4 species (1.06%). It 

includes 3 Balkan endemites – Achillea clypeolata 

Sibth. et Sm., Aesculus hippocastanum L., Inula 

aschersoniana Janka and 1 Balkan subendemite – 

Syringa vulgaris L. 

Table 5. Floristic elements 

Floristic elements Species 

Circumboreal type 136 

European type 101 

Mediterranean type 67 

Pontic type 27 

Adventive type 20 

Cosmopolitan type 19 

Balkan endemic and 

subendemic type 

4 

Other 2 

This distribution can be explained by the location 

of the plateau in the transcontinental climate region. 

The proximity of the plateau to the border of a 

48 

19 5 13 

11 

a 

a-b 

a-p 

b 

b-p 

p 

sh 

sh-t 

t


temperate region is the reason for the prevalence of 

circumboreal and European floristic elements. At the 

same time, the impact of the continentalmediterranean 

region in terms of the Black Sea and 

the karst topography create conditions for the 

development of a large number of mediterranean 

species. 

The flora of the plateau includes significant 

number of relict species: 29. They account for 7.71% 

of the total number of species. The majority of the 

relict species are Tertiary relicts. They are 28 species: 

Abies alba Mil., Acer campestre L., Acer 

pseudoplatanus L., Acer tataricum L., Aesculus 

hippocastanum L., Betula pendula Roth, Carpinus 

betulus L., Carpinus orientalis Mill., Celtis australis 

L., Cercis siliquastrum L., Clematis vitalba L., 

Corylus avellana L., Cotinus coggygria Scop., 

Fraxinus excelsior L., Fraxinus ornus L., Hedera 

helix L., Juniperus communis L., Picea abies (L.) 

Karsten, Pinus nigra Arn., Populus alba L., Populus 

nigra L., Ruscus aculeatus L., Salix alba L., Salix 

caprea L., Smilax excelsa L., Taxus baccata L., 

Ulmus minor Mill., Viscum album L. One of the relict 

species is quaternary – Galanthus nivalis L. 

39 species with protection statute are described. 

One of them – Himantoglossum caprinum (Bieb.) C. 

Koch., is included in the list of species, protected by 

the Berne Convention and Natura 2000. In CITES 10 

species are included: Adonis vernalis L., Anacamptis 

pyramidalis C. Rich., Galanthus elwesii Hook. fil., 

Galanthus nivalis L., Himantoglossum caprinum 

(Bieb.) C. Koch., Orchis morio L., Orchis purpurea 

Huds., Orchis simia L., Orchis tridentata Scop., 

Platanthera chlorantha (Cust.) Rchb. In the IUCN 

Red List for Bulgaria 5 species are included under the 

category „Threatened”: Aesculus hippocastanum L., 

Galanthus elwesii Hook. fil., Galanthus nivalis L., 

Juniperus sabina L., Taxus baccata L., 2 species are 

included under the category „Vulnerable”: 

Anacamptis pyramidalis C. Rich., Himantoglossum 

caprinum (Bieb.) C. Koch, 2 species are in the 

category „Nearly threatened”: Anemone sylvestris L., 

Cercis siliquastrum L. and 1 species is included in 

the category „Of least concern”: Tilia rubra DC. In 

the Red book for Bulgaria 4 species are included in 

the category „Endangered”: Aesculus hippocastanum 

L., Anemone sylvestris L., Galanthus nivalis L., 

Taxus baccata L. and 4 species are included in the 

21 

category „Rare”: Artemisia pontica L., Cercis 

siliquastrum L., Juniperus sabina L., Tilia rubra DC. 

In the Biological Diversity Act 8 species are included 

in the category „Protected”: Aesculus hippocastanum 

L., Anacamptis pyramidalis C. Rich., Anemone 

sylvestris L., Galanthus elwesii Hook. fil., Galanthus 

nivalis L., Himantoglossum caprinum (Bieb.) C. 

Koch., Juniperus sabina L., Taxus baccata L. In the 

category “Under the protection and regulated use of 

nature” are 14 species: Asparagus officinalis L., 

Crocus pallasii Bieb., Echinops sphaerocephalos L., 

Gypsophila paniculata L., Helichrysum areanrium 

(L.) Moench., Lilium martagon L., Orchis morio L., 

Orchis purpurea Huds., Orchis simia L., Orchis 

tridentata Scop., Polygonatum odoratum (Mill.) 

Druce, Ruscus aculeatus L., Salix caprea L., Scilla 

bifolia L. Collecting herbs is prohibited from the 

natural habitats of 15 species: Adonis vernalis L., 

Althaea officinalis L., Artemisia santonicum L., 

Asarum europaeum L., Asplenium trichomanes L., 

Convallaria majalis L., Glaucium flavum Crantz, 

Helichrysum areanrium (L.) Moench., Orchis morio 

L., Orchis purpurea Huds., Orchis simia L., Orchis 

tridentata Scop., Phyllitis scolopendrium (L.) 

Newm., Ruscus aculeatus L., Valeriana officinalis L. 

Under a restrictive regime are 4 species: Berberis 

vulgaris L., Carlina acanthifolia All., Galium 

odoratum (L.) Scop., Sedum acre L. 

The anthropophytes among the medicinal plants 

are 236 species (62.77%). Many of them are 

considered weed or ruderal plants. The most common 

as weed are: Anagallis arvensis L., Brassica nigra 

(L.) Koch, Centaurea cyanus L., Chenopodium 

album L., Chenopodium polyspermum L., Consolida 

hispanica (Costa) Greut. et Burdet, Consolida regalis 

S. F. Gray, Cynodon dactylon (L.) Pers., Datura 

stramonium L., Myosotis arvensis (L.) Hill, Nigella 

arvensis L., Papaver rhoeas L., Senecio vulgaris L., 

Stellaria media (L.) Vill., Thlaspi arvense L., 

Xanthium strumarium L. Оf the ruderal plants most 

common are: Capsella bursa-pastoris Moench., 

Cardaria draba (L.) Desv., Chamomilla recutita (L.) 

Rausch., Chelidonium majus L., Conium maculatum 

L., Conyza canadensis (L.) Cronq., Heracleum 

sibiricum L., Lactuca serriola L., Parietaria 

lusitanica L., Sambucus ebulus L., Solanum 

dulcamara L., Urtica dioica L.


4. Conclusions 

[3] KOPRALEV, I. (main ed.), 2002. Geography of 

Bulgaria. Physical and socio-economic 

Considerable taxonomical diversity of the geography, Institute of Geography, BAS, 

medicinal plants of Provadiisko Plateau is identified: Farkom, Sofia, 760 pp. 

376 species of vascular plants from 261 genera and [4] NINOV, N., 2002. Soils, in Kopralev, I. (main 

86 families. 

ed.). Geography of Bulgaria. Physical and socioeconomic 

geography, Institute of Geography, 

BAS, Farkom, Sofia, 760 pp. 

Most families (77.91%) and genera (98.85%) are 

represented in small numbers: 1 to 4. 

The analysis of life forms indicates the 

predominnce of geophytes, followed by the groups 

phanerophytes and hemi cryptophytes. 

The biological types are represented mainly by 

perennial herbaceous plants (53.19%) and annual 

herbaceous plants (12.77%). 

The identified medicinal plants can be 

categorized into 7 types of floristic elements. The 

highest percentage species are of the circumboreal 

type (36.17%). 

Among the medicinal plants of Provadiisko 

Plateau 4 endemites and 29 relicts are described. 

39 species with protection status are described: 

the use of 1 species is restricted by the Berne 

Convention and Natura 2000; 10 species are included 

in CITES; 10 species are included in IUCN Red List 

for Bulgaria; 8 species appear in the Red book for 

Bulgaria; 22 species are included in the Biological 

Diversity Act; 14 species are included in the category 

“Under the protection and regulated use of nature”, 

the collecting of herbs from their natural habitats is 

prohibited for 15 species, and 4 species are under a 

restrictive regime. 

The anthropophytes among the medicinal plants 

are 236 species (62.77%). Many of them are 

considered weed or ruderal plants. 

5. References 

[1] GALABOV, J., 1966. Main lines of the relief 

(Common morphographic and morphometric 

characteristics), in Geography of Bulgaria, 

Physical geography – Relief, Vol. 1, Sofia. 

[2] VELEV, S., 2002. Climatic zoning, in Kopralev, 

I. (main ed.). Geography of Bulgaria. Physical 

and socio-economic geography, Institute of 

Geography, BAS, Farkom, Sofia, 760 pp. 

22 

[5] BONDEV, I., 1991. The vegetation of Bulgaria. 

Map in М 1:600 000 with explanatory text, 

University Press St. Kliment Ohridski, Sofia, 183 

pp. 

[6] STANEV, S., 2001. Little known names from 

Bulgarian botany, Pensoft, Sofia – Moscow, 202 

pp. 

[7] VELENOVSKY, J., 1891. Flora Bulgarica, Praga, 

676 рp. 

[8] VELENOVSKY, J., 1898. Flora Bulgarica, 

Supplementum I, Praga, 420 рp. 

[9] ZAHARIEV, D., Dimitrov D., 2009. The 

medicinal plants in area of Provadiisko Plato 

(Municipality Provadia), 8th National conference 

with international participation „Natural sciences 

– 2009”, 2-3.10.2009, Varna (upcoming). 

[10] ZAHARIEV, D., Uzunov G., 2009. A study of 

the flora in Protected place Madarski skalni 

venci, 8th National conference with international 

participation „Natural sciences – 2009”, 2- 

3.10.2009, Varna (upcoming). 

[11] Council Directive 92/43/EEC of the European 

Community to protect natural habitats and of 

wild fauna and flora. 

[12] Flora of PR Bulgaria, Vol. І-Х, 1963-1995, 

Publishing House of BAS, Sofia. 

[13] CHASE, M. (corresponding author), 2003. An 

update of the Angiosperm Phylogeny Group 

classification for the orders and families of 

flowering plants: APG II, The Linnean Society of 

London, Botanical Journal of the Linnean 

Society, 141: 399–436. 

[14] PAVLOV, D., 2006. Phytocoenology, 

Publishing House of University of Forestry, 

Sofia, 251 pp. 

[15] KOZUHAROV, S. (ed.), 1992. Identifier of the 

vascular plants in Bulgatia, Nauka i izkustvo, 

Sofia, 788 pp.


[16] ASIOV B., Petrova A., Dimitrov D., Vasilev R., 

2006. Conspectus of the Bulgarian vascular flora. 

Distribution maps and floristic elements, 

Bulgarian Biodiversity Foundation, Sofia, 452 

pp. 

[17] GRUEV, B., Kuzmanov B., 1994 – General 

biogeography, University Press St. Kliment 

Ohridski, Sofia, 498 pp. 

[18] PEEV, D., 2001. National park Rila. 

Management plan 2001 – 2010. Adopted by 

Resolution №522 of Council of Ministers on 

04.07.2001, Sofia, 338 pp. 

[19] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić 

D., 2005. Flora “Rimskog šanca” (Vojvodina, 

Srbija), 8th Symposium on the flora of 

Southeastern Serbia and Neighbouring Regions, 

Niš, 20-24.06.2005, Abstracts, рр. 55. 

[20] PEEV, D., Kozuharov S., Anchev M., Petrova 

A., Ivanova D., Tzoneva S., 1998. Biodiversity 

of Vascular Plants in Bulgaria, In: Curt Meine 

(ed.), Bulgaria's Biological Diversity: 

Conservation Status and Needs Assessment, 

Volumes I and II, Washington, D.C., 

Biodiversity Support Program, pp. 55–88. 

[21] Convention on International Trade in 

Endangered Species of Wild Fauna and Flora, 

State Gazette number 6 from 21 Januari 1992. 

[22] Red book of PR Bulgaria, Vol. 1, Plants, 1984, 

Publishing House of BAS, Sofia, 447 pp. 

[23] PETROVA А., Vladimirov V. (eds.), 2009. Red 

List of Bulgarian vascular plants, Phytologia 

Balcanica 15 (1): 63–94. 

[24] Biological Diversity Act, State Gazette number 

77 from 9 august 2002, pp. 9–42. Amended in 

State Gazette number 94 from 16 November 

2007. 

[25] Order number RD-72 from 3 februari 2006 for 

special arrangements for the conservation and 

use of medicinal plants, State Gazette number 16 

from 21 Februari 2006. 

[26] STEFANOV, B., Kitanov B., 1962. Kultigenen 

plants and kultigenen vegetation in Bulgaria, 


23


THE PLANTS WITH PROTECTION STATUTE, ENDEMITES AND RELICTS 

OF THE SHUMENSKO PLATEAU 

Dimcho ZAHARIEV, Elka RADOSLAVOVA 


115 Universitetska Str., 9712, Shumen, Bulgaria 

dimtchoz@yahoo.com 

__________________________________________________________________________________________ 

Abstract: As a result of our investigations of the Shoumen Plateau in the period 1998-2009, 786 species were 

identified, of which the number of species with conservation status is 80 (10.18%). 2 of those species are 

included in Appendix II of Directive 92/43/ЕЕС. 24 of the species are included in CITES. 32 species are 

included in the IUCN Red List for Bulgaria under the following categories: threatened – 13, vulnerable – 9, 

nearly threatened – 5 and least concern – 5 species. In the Red book for Bulgaria, there are 7 endangered species 

and 14 are rare plants. In the Biological Diversity Act, 23 species are included in Appendix 3 and further 28 

species – in Appendix 4. The collecting of herbs from their natural habitats is prohibited for 12 species, and 6 

species are under a restriction. 29 species (3.69%) are endemites. These are 17 Balkan subendemites, 9 Balkan 

endemites and 3 Bulgarian endemites. The flora of the plateau includes a significant number of relict species – 

42. (5.34%). The majority of them, 39 species, are Tertiary relicts, 2 are quaternary relicts and 1 is a postglacial 

steppe relict. 

Keywords: Shumensko Plateau, plants with protection statute, endemites, relicts. 

__________________________________________________________________________________________ 


Shumensko Plateau refers to an area in the hills 

east of the Danube plain, which was declared 

protected by Natura 2000. This was determined by the 

hills’ role in support of the biodiversity among large 

territories of scattered forests. The majority of the 

Shumensko Plateau area – 3929.9 ha (53%), was 

declared for National Park in 1980. In 2003, the park 

was recognized as Nature Park. The regime of use 

and management of the park is determined by the 

Protected Areas Act [1] and the Management Plan for 

the Nature Park [2]. 

In the park is located the Bukaka Preserve. This 

is a forest area of 63.04 ha, declared protected due to 

the indigenous forest that has existed there for several 

centuries and is comprised of Fagus sylvatica subsp. 

moesiaca. On the territory of the preserve, all human 

activity is prohibited, except for people passing on 

specifically marked paths. 

Shumensko Plateau has been declared protected 

by Natura 2000 and its estimated area is 4490.62 hа. 

This territory is also protected under the Council 

Directive 92/43/EEC of the European Community 

for protecting natural habitats of wild fauna and flora 

[3]. 

The unique combination of conditions in terms 

of topography, water resources, climate and soil, 

determine the diversity of the plant species in the 

area. In the past, Velenovsky and his collaborators 

Hermengild Shkorpil and Anani Iavashev began the 

study of the plateau’s flora. In the 1980s, they 

collected the first botanical data in Northeast 

Bulgaria, including the area of the Shumen vicinity 

[4]. Their research is presented in the first volume on 

the Bulgarian flora [5] and its supplement [6]. 

Davidov [7] conducted his own research on the flora 

of Shumen and the territory around the town. Further 

information about individual species, distributed on 

the plateau, can be found in Stoyanov and Stefanov 

[8, 9, 10], Stoyanov, Stefanov and Kitanov [11] and 

in Flora of PR Bulgaria, Vol. І – Х [12]. The 

diversity of species of the Orchidaceae family has 

been studied by Radoslavova [13]. In the 

Management Plan for the National Park Shumensko 

Plateau [2]: there are 550 species of vascular plants 


The plants with protection statute, endemites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) 

(i.e. mosses not counted) described in that source. Our 

studies [14] show that the number of vascular plants 

on the territory of the entire plateau is 786 species. 

According to the forest development project of 

the Shumen Forestry [15], a total of 16 species have 

conservation statute and are included in the Red book 

of PR Bulgaria [16]. Seven of the species are 

endangered: Aesculus hippocastanum L., Anacamptis 

pyramidalis C. Rich., Anemone sylvestris L., 

Castanea sativa Mill., Galanthus nivalis L., 

Himantoglossum hircinum (L.) Spreng., Paeonia 

tenuifolia L. Nine of the species are rare: Atropa 

belladonna L., Celtis caucasica Willd., Cercis 

siliquastrum L., Cyclamen coum Mill., Fibigia 

clypeata (L.) Medic., Fritillaria pontica Wahl., 

Haplophyllum thesioides G. Don., Jurinea ledeborii 

Bunge., Pastinaca umbrosa Stev. ex DC. Six of these 

species are protected by the Biological Diversity Act 

(BDA) [17]: Aesculus hippocastanum L., Anacamptis 

pyramidalis C. Rich., Anemone sylvestris L., 

Cyclamen coum Mill., Galanthus nivalis L., 

Himantoglossum hircinum (L.) Spreng. 

Six of the plateau species are listed in the Red 

Book of the district Shumen [18]. Two of them are 

endangered: Lilium martagon L. and Campanula 

euxina (Vel.) Ancev. Four of the species are rare: 

Himantoglossum hircinum (L.) Spreng., Anacamptis 

piramidalis (L.) Rich., Ruscus hyppoglosum L. and 

Galium paschale Forsskal. 

In the Management Plan of the National Park 

Shumensko Plateau [2] are found 18 species with 

conservation statute that are also listed in the Red 

book of PR Bulgaria. Five of them are in the category 

“endangered”: Anemone sylvestris L., Colchicum 

davidovii Stefanov, Galanthus nivalis L., Ruta 

graveolens L., Veronica spicata L. Thirteen species 

fall into the category “rare”: Anthemis regis-borisii 

Stoj. et Acht., Anthemis rumelica (Velen.) Stoj. et 

Acht., Celtis caucasica Willd., Cyclamen coum Mill., 

Erodium hoefftianum C. A. Meyer, Fibigia clypeata 

(L.) Medic., Fritillaria graeca Boiss. & Spruner, 

Fritillaria pontica Wahl., Galium bulgaricum Vel., 

Haplophyllum thesioides G. Don., Hedysarum 

tauricum Pallas ex Willd., Jurinea ledeborii Bunge., 

Pastinaca umbrosa Stev. ex DC. 


26 

Our study of the flora of the Shoumen Plateau 

was conducted on the route method in 1998 – 2009. 

The names of the taxons are taken from the Flora of 

PR Bulgaria, Vol. І – Х [12]. The update of the 

taxons is consistent with APG II [19]. 

The endemites are represented by Asiov et all. 

[20]. 

The relicts are represented by Gruev and 

Kuzmanov [21], Peev [22], Boža et all. [23], Peev et 

all. [24]. 

The conservation statute is recognized using the 

following documents: Council Directive 92/43/EEC 

of the European Community to protect natural 

habitats and of wild fauna and flora [3], Convention 

on International Trade in Endangered Species of 

Wild Fauna and Flora (CITES) [25], Red book of PR 

Bulgaria [16], IUCN Red List for Bulgaria [26], 

Biological Diversity Act [17], Order for special 


medicinal plants [27]. 

3. Results and Discussion 

The analysis of the received data leads to the 

following results and conclusions: Two species, 

Anacamptis pyramidalis C. Rich. and 

Himantoglossum hircinum (L.) Spreng., of the 16 

protected and listed as endangered species in the 

forest development project of the Shumen Forestry 

do not fall into any category protected by the Red 

Book of PR Bulgaria. They are listed as “rare” in the 

Red Book of the district Shumen. Furthermore, in the 

Red List of the Bulgarian vascular plants, they are 

given similar status – “ vulnerable”. Three of the 

species: Atropa belladonna L., Castanea sativa Mill. 

and Paeonia tenuifolia L. we did not find on the 

territory of the plateau. Himantoglossum hircinum 

(L.) Spreng. is incorrectly recorded as located in 

Bulgaria and should be replaced with the correct 

species name, Himantoglossum caprinum (Bieb.) C. 

Koch. The name Celtis caucasica Willd. is obsolete, 

now replaced by Celtis glabrata Steven. 

As a result of several years of observations, we 

found that populations of the following species have 

increased: Anacamptis pyramidalis C. Rich., 

Cyclamen coum Mill., Galanthus nivalis L., 

Himantoglossum caprinum (Bieb.) C. Koch., Lilium 

martagon L. and Ruta graveolens L. Therefore, they 

are not really endangered anymore.

Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010) 

From the 18 protected species included in the 

Management Plan of National Park Shumensko 

Plateau, 2 species, Colchicum davidovii Stefanov and 

Veronica spicata L., listed as endangered, have not 

been confirmed by us as existing on the plateau. We 

think that Colchicum davidovii Stefanov has 

disappeared from the flora of the plateau. 

Four species listed as rare or “rare” species, we 

did not found on the plateau: Anthemis rumelica 

(Velen.) Stoj. et Acht., Fritillaria graeca Boiss. & 

Spruner, Galium bulgaricum Vel., Hedysarum 

tauricum Pallas ex Willd. 

The new data for the conservation statute of the 

species, established by us within the realm of the 

Shumensko Plateau, shows the following: 

The total number of species with conservation 

statute is 80 (Figure 1). This is a 10.18% from the 

total number of species found on the Shumensko 

Plateau. We found the following species: 

1. Aegilops geniculata Roth 

2. Aesculus hippocastanum L. 

3. Althaea officinalis L. 

4. Anacamptis pyramidalis C. Rich. 

5. Anemone sylvestris L. 

6. Anthemis regis-borisii Stoj. et Acht. 

7. Artemisia pedemontana Balb. 

8. Asarum europaeum L. 

9. Asparagus tenuifolius Lam. 

10. Asparagus verticillatus L. 

11. Asplenium trichomanes L. 

12. Berberis vulgaris L. 

13. Betonica officinalis L. 

14. Bupleurum affine Sadl. 

15. Bupleurum apiculatum Friv. 

16. Bupleurum praealtum L. 

17. Bupleurum rotundifolium L. 

18. Campanula euxina (Vel.) Ancev 

19. Carlina acanthifolia All. 

20. Celtis glabrata Steven 

21. Centaurea marshalliana Spreng. 

22. Cephalanthera damasonium (Mill.) Druce 

23. Cephalanthera longifolia (L.) Fritsch 

24. Cephalanthera rubra (L.) Rich. 

25. Cercis siliquastrum L. 

26. Convallaria majalis L. 

27. Crocus flavus West. 

28. Crocus pallasii Bieb. 

29. Cyclamen coum Mill. 

30. Dactylorhiza saccifera (Brongn.) Soo 

27 

31. Dryopteris filix-mas (L.) Schott 

32. Echinops sphaerocephalos L. 

33. Epipactis helleborine (L.) Crantz 

34. Epipactis microphylla (Ehrh.) Sw. 

35. Epipactis purpurata Smith 

36. Erodium hoefftianum C. A. Mey. 

37. Fibigia clypeata (L.) Medic. 

38. Fritillaria pontica Wahl. 

39. Galanthus elwesii Hook. fil. 

40. Galanthus nivalis L. 

41. Galium odoratum (L.) Scop. 

42. Galium rubioides L. 

43. Gypsophila paniculata L. 

44. Haplophyllum thesioides G. Don. 

45. Helichrysum arenarium (L.) Mornh. 

46. Himantoglossum caprinum (Bieb.) C. Koch 

47. Juniperus sabina L. 

48. Jurinea ledebourii Bunge 

49. Lilium martagon L. 

50. Limodorum abortivum (L.) Sw. 

51. Listera ovata (L.) R. Br. 

52. Neottia nidus-avis (L.) Rich. 

53. Ophrys apifera Huds. 

54. Ophrys cornuta Stev. 

55. Ophrys mammosa Desf. 

56. Orchis morio L. 

57. Orchis purpurea Huds. 

58. Orchis simia Lam. 

59. Orchis tridentata Scop. 

60. Pastinaca umbrosa Stev. et DC. 

61. Phyllitis scolopendrium (L.) Newm. 

62. Platanthera chlorantha (Cust.) Rchb. 

63. Polygonatum odoratum (Mill.) Druce 

64. Polystichum aculeatum (L.) Roth 

65. Primula veris L. 

66. Pulmonaria mollis Horn. 

67. Ruscus aculeatus L. 

68. Ruscus hypoglossum L. 

69. Ruta graveolens L. 

70. Salix caprea L. 

71. Scilla bifolia L. 

72. Sedum acre L. 

73. Sternbergia colchiciflora Waldst. et Kit. 

74. Stipa capillata L. 

75. Stipa pulcherrima C. Koch 

76. Stipa tirsa Stev. 

77. Taxus baccata L. 

78. Tilia rubra DC. 

79. Valeriana officinalis L.


80. Vicia pisiformis L. 

Two species are included in Application II of 

Directive 92/43/ЕЕС: Cyclamen coum Mill. and 

Himantoglossum caprinum (Bieb.) C. Koch. 

60 

50 

40 

30 

20 

10 

0 

2 

24 

32 

21 

51 

1 

12 

6 

29 

42 

Directive 92/43/ЕЕС 

CITES 

IUCN Red List 

Red book 

BDA 

Herbs prohibited from collecting 

Herbs in the restrictive regime 

Endemites 

Relicts 

Fig. 1. Proportion of species with 

conservation status, endemites and relicts 

In the Convention on International Trade in 

Endangered Species of Wild Fauna and Flora 

(CITES) are included 24 species: Anacamptis 

pyramidalis C. Rich., Cephalanthera damasonium 

(Mill.) Druce, Cephalanthera longifolia (L.) Fritsch, 

Cephalanthera rubra (L.) Rich., Cyclamen coum 

Mill., Dactylorhiza saccifera (Brongn.) Soo, 

Epipactis helleborine (L.) Crantz, Epipactis 

microphylla (Ehrh.) Sw., Epipactis purpurata Smith, 

Galanthus elwesii Hook. fil., Galanthus nivalis L., 

Himantoglossum caprinum (Bieb.) C. Koch, 

Limodorum abortivum (L.) Sw., Listera ovata (L.) R. 

Br., Neottia nidus-avis (L.) Rich., Ophrys apifera 

Huds., Ophrys cornuta Stev., Ophrys mammosa 

Desf., Orchis morio L., Orchis purpurea Huds., 

Orchis simia Lam., Orchis tridentata Scop., 

Platanthera chlorantha (Cust.) Rchb., Sternbergia 

colchiciflora Waldst. et Kit. 

The IUCN Red List for Bulgaria are included 32 

species. In category „threatened” are included 13 

species: Aesculus hippocastanum L., Anthemis regisborisii 

Stoj. et Acht., Artemisia pedemontana Balb., 

Campanula euxina (Vel.) Ancev, Celtis glabrata 

Steven, Epipactis purpurata Smith, Galanthus elwesii 

Hook. fil., Galanthus nivalis L., Juniperus sabina L., 

Jurinea ledebourii Bunge, Ophrys apifera Huds., 

Ruta graveolens L., Taxus baccata L. In category 

„vulnerable” are included 9 species: Anacamptis 

pyramidalis C. Rich., Epipactis microphylla (Ehrh.) 

Sw., Fibigia clypeata (L.) Medic., Haplophyllum 

28 

thesioides G. Don., Himantoglossum caprinum 

(Bieb.) C. Koch, Limodorum abortivum (L.) Sw., 

Ophrys cornuta Stev., Ophrys mammosa Desf., 

Pastinaca umbrosa Stev. et DC. In category „nearly 

threatened” 5 species: Anemone sylvestris L., Cercis 

siliquastrum L., Erodium hoefftianum C. A. Mey., 

Galium rubioides L., Vicia pisiformis L. In category 

„least concern” are included 5 species: Aegilops 

geniculata Roth, Cyclamen coum Mill., Fritillaria 

pontica Wahl., Pulmonaria mollis Horn., Tilia rubra 

DC. 

In the Red book for PR Bulgaria are included 

total of 21 species. In the category „endangered” are 

included 7 species: Aesculus hippocastanum L., 

Anemone sylvestris L., Artemisia pedemontana 

Balb., Galanthus nivalis L., Galium rubioides L., 

Ruta graveolens L., Taxus baccata L. In category 

„rare” are included 14 species: Anthemis regisborisii 

Stoj. et Acht., Celtis glabrata Steven, Cercis 

siliquastrum L., Cyclamen coum Mill., Erodium 

hoefftianum C. A. Mey., Fibigia clypeata (L.) 

Medic., Fritillaria pontica Wahl., Haplophyllum 

thesioides G. Don., Juniperus sabina L., Jurinea 

ledebourii Bunge, Limodorum abortivum (L.) Sw., 

Pastinaca umbrosa Stev. et DC., Tilia rubra DC., 

Vicia pisiformis L. 

In the Biological Diversity Act are included total 

of 51 species. In the category „protected” 

(Application 3) are included 23 species: Aesculus 

hippocastanum L., Anacamptis pyramidalis C. Rich., 

Anemone sylvestris L., Anthemis regis-borisii Stoj. et 

Acht., Artemisia pedemontana Balb., Campanula 

euxina (Vel.) Ancev, Centaurea marshalliana 

Spreng., Cyclamen coum Mill., Epipactis purpurata 

Smith, Fritillaria pontica Wahl., Galanthus elwesii 

Hook. fil., Galanthus nivalis L., Galium rubioides 

L., Haplophyllum thesioides G. Don., 

Himantoglossum caprinum (Bieb.) C. Koch, 

Juniperus sabina L., Jurinea ledebourii Bunge, 

Limodorum abortivum (L.) Sw., Ophrys apifera 

Huds., Ophrys cornuta Stev., Ophrys mammosa 

Desf., Ruta graveolens L., Taxus baccata L. 

In the category “under protection and under 

controlled use” (Application 4) are 28 species: 

Asparagus tenuifolius Lam., Asparagus verticillatus 

L., Bupleurum affine Sadl., Bupleurum apiculatum 

Friv., Bupleurum praealtum L., Bupleurum 

rotundifolium L., Crocus flavus West., Crocus 

pallasii Bieb., Dactylorhiza saccifera (Brongn.) Soo,


Dryopteris filix-mas (L.) Schott, Echinops 

sphaerocephalos L., Gypsophila paniculata L., 

Helichrysum arenarium (L.) Mornh., Lilium 

martagon L., Orchis morio L., Orchis purpurea 

Huds., Orchis simia Lam., Orchis tridentata Scop., 

Polygonatum odoratum (Mill.) Druce, Polystichum 

aculeatum (L.) Roth, Primula veris L., Ruscus 

aculeatus L., Ruscus hypoglossum L., Salix caprea 

L., Scilla bifolia L., Stipa capillata L., Stipa 

pulcherrima C. Koch, Stipa tirsa Stev. 

Prohibited is the collecting ofherbs from the 

natural habitats of 12 species: Althaea officinalis L., 

Asarum europaeum L., Asplenium trichomanes L., 

Convallaria majalis L., Helichrysum arenarium (L.) 

Mornh., Orchis morio L., Orchis purpurea Huds., 

Orchis simia Lam., Orchis tridentata Scop., Phyllitis 

scolopendrium (L.) Newm., Ruscus aculeatus L., 

Valeriana officinalis L. 

Under a controlled use are 6 species: Berberis 

vulgaris L., Betonica officinalis L., Carlina 

acanthifolia All., Galium odoratum (L.) Scop., 

Primula veris L., Sedum acre L. 

Endemic species (Figure 1) are relatively well 

represented – 29 species (3.69% of all species on the 

plateau). Their number is close to the nation-wide 

average – 4.86% [24]. This group includes 17 Balkan 

subendemites: Campanula grossekii Heuff., 

Campanula lingulata W. et K., Carduus candicans 

Waldst. et Kit., Chaerophyllum byzantinum Boiss., 

Doronicum orientale Hoffm., Galium heldreichii 

Hal., Galium paschale Forsskal, Galium 

pseudoaristatum Schur., Ophrys cornuta Stev., 

Pseudolysimachion barrelieri (Schott ex Roem. et 

Schult.) Holub, Salvia amplexicaulis Lam., Senecio 

papposus (Reichenb.) Less., Stachys obliqua Waldst. 

et Kit., Symphytum ottomanum Friv., Syringa vulgaris 

L., Thesium simplex Vel., Verbascum lychnitis L. The 

Balkan endemites are 9 species: Achillea clypeolata 

Sibth. et Sm., Aesculus hippocastanum L., Bupleurum 

apiculatum Friv., Inula aschersoniana Janka, Knautia 

macedonica Griseb., Koeleria simonkaii Adam., 

Onosma thracica Vel., Salvia ringens Sibth. et Sm., 

Sesleria latifolia (Adam.) Deg. The Bulgarian 

endemites are 3 species: Anthemis regis-borisii Stoj. 

et Acht., Campanula euxina (Vel.) Ancev, Myosotis 

aspera Vel. 

Data for the relict species on the area of the 

plateau was first published by Zahariev and 

Radoslavova [14]. The flora of the plateau included 

29 

significant number of relict species – 42 (Figure 1). 

They account for 5.34% of the total species. The 

majority of them, 39 species, are Tertiary relicts: 

Abies alba Mil., Acer campestre L., Acer hyrcanum 

Fisch. et C. A. Meyer, Acer pseudoplatanus L., Acer 

tataricum L., Aesculus hippocastanum L., Betula 

pendula Roth, Carpinus betulus L., Carpinus 

orientalis Mill., Celtis glabrata Steven, Cercis 

siliquastrum L., Clematis vitalba L., Corylus 

avellana L., Cotinus coggygria Scop., Cyclamen 

coum Mill., Fraxinus excelsior L., Fraxinus ornus 

L., Hedera helix L., Juniperus communis L., 

Lathyrus aureus (Stev.) Brandza, Pastinaca umbrosa 

Stev. et DС., Phragmites australis (Cav.) Steud., 

Picea abies (L.) Karsten, Pinus nigra Arn., Populus 

alba L., Populus nigra L., Populus tremula L., 

Pteridium aquilinum (L.) Kuhn., Quercus cerris L., 

Quercus dalechampii Ten., Ruscus aculeatus L., 

Ruscus hypoglossum L., Salix alba L., Salix caprea 

L., Taxus baccata L., Ulmus laevis Pall., Ulmus 

minor Mill., Viburnum lantana L., Viscum album L. 

They were widespread during the Tertiary, but their 

habitats today are much smaller. 

The second group are quaternary relicts. They 

have become part of our flora as a result of glaciation 

during the Quaternary. Therefore, they are 

considered glacial relicts. On the plateau, there are 

two such species: Limodorum abortivum (L.) Sw. 

and Galanthus nivalis L. From the third group, the 

postglacial steppe relict, only one species is found: 

Sternbergia colchiciflora Waldst. et Kit. 

The species with highest conservation value, i.e. 

those that fall into the categories of being 

endangered and vulnerable, are 24 in number. 

With the highest conservation value is Cyclamen 

coum Mill., which is included in 6 different lists of 

endangered species: Directive 92/43/ЕЕС, CITES, 

IUCN Red List, Red book, BDA, Tertiary relicts. 

Second comes the group of the species 

Galanthus nivalis L. and Limodorum abortivum (L.) 

Sw. They appear in 5 different lists: CITES, IUCN 

Red List, Red book, BDA, quaternary relicts. This 

also applies to Aesculus hippocastanum L., which is 

included in the following lists: IUCN Red List, Red 

book, BDA, Balkan endemites, Tertiary relicts. 

The third group of species that is listed in 4 lists 

is Himantoglossum caprinum (Bieb.) C. Koch. 

(Directive 92/43/ЕЕС, CITES, IUCN Red List, ЗБР), 

Anthemis regis-borisii Stoj. et Acht. (IUCN Red List,


Red book, BDA, Bulgarian endemites), Ophrys 

cornuta Stev. (CITES, IUCN Red List, BDA, Balkan 

subendemites), Taxus baccata L. (IUCN Red List, 

Red book, BDA, Tertiary relicts). 

The largest is the group of species that appear in 

the following lists: 

• CITES, IUCN Red List, BDA – Anacamptis 

pyramidalis C. Rich., Epipactis purpurata 

Smith, Galanthus elwesii Hook. fil., Ophrys 

apifera Huds., Ophrys mammosa Desf.; 

• IUCN Red List, Red book, BDA – Anemone 

sylvestris L., Fritillaria pontica Wahl., Galium 

rubioides L., Haplophyllum thesioides G. 

Don., Juniperus sabina L., Jurinea ledebourii 

Bunge, Ruta graveolens L.; 

• IUCN Red List, Red book, Tertiary relicts – 

Celtis glabrata Steven, Cercis siliquastrum L., 

Pastinaca umbrosa Stev. et DC.; 

• IUCN Red List, BDA, Bulgarian endemites – 

Campanula euxina (Vel.) Ancev. 


The total number of species with conservation 

statute that we found on the Shoumen plateau is 80 

(10.18% of all species on the plateau). It is 

significantly larger than the data published by other 

authors. In our study, we use more recent documents 

on nature conservation. They total 6 in comparison to 

3 or 4 in previous publications. The species that we 

described generally appear in 12 lists of endangered 

species. 

The endemic species that we found on the plateau 

and described are 29 species (3.69% of the total 

number of species). They include 17 Balkan 

subendemites, 9 Balkan endemites and 3 Bulgarian 

endemites. 

The flora of the plateau includes significant 

number of relict species – 42 (5.34% of the total 

number of species). The majority of them are Tertiary 

relicts: 39 species, 2 are quaternary relicts and 1 is 

postglacial steppe relict. 

The largest number of species of conservation 

statute confirms the importance of the Shoumen 

Plateau as a protected site, preserving the wellbeing 

of nature in the future. 

30 

5. References 

[1] Protected Areas Act, State Gazette number 133 

from 11 November 1998, Amended in State 

Gazette number 98 from 12 November 1999,..., 

Amended in State Gazette number 19 from 13 

March 2009. 

[2] ANDREEV, N., 1992. Botanical characteristics 

of National Park Shumensko Plateau, in 

National Park Shumensko Plateau. Technical 

Project Green Construction, Agrolesproject, pp. 

17–62. 




[4] STANEV, S., 2001. Little known names from 

Bulgarian botany, Pensoft, Sofia – Moscow, 

202 pp. 


Praga, 676 рp. 


Supplementum I, Praga, 420 рp. 

[7] DAVIDOV, B., 1904. Contribution to study the 

flora of the district of Shumen, Sbornik ot 

narodni umotvoreniya, ХХ (II): 1–54. 

[8] STOIANOV, N., Stefanov B., 1924-1925. Flora 

of Bulgaria, Vol. I-II, Sofia, pp. 1367. 


of Bulgaria, Vol. I-II, Sofia. 


of Bulgaria, Vol. I-II, Sofia, pp. 1361. 

[11] STOIANOV, N., Stefanov B., Kitanov, B., 

1966-1967. Flora of Bulgaria, Vol. I-II, Nauka i 

izkustvo, Sofia, pp. 1325. 



[13] RADOSLAVOVA, Е., 2002. The Orchids of the 

Shumensko Plateau, Snejanka Petkova – AR, 

Shumen, pp. 48. 

[14] ZAHARIEV, D., Radoslavova, E., 2010. The 

Plants of the Shumensko Plateau, Himera, 

Shumen, pp. 597. 

[15] Forest development project of the Shumen State 

Forestry, district Shumen, Vol. I, 2002, 

Anemone Ltd., Sofia, pp. 148. 




77 from 9 august 2002, pp. 9–42. Amended in


State Gazette number 94 from 16 November 

2007. 

[18] BESHKOV, V. et all.. (eds.), 1994. The Red 

book of the district Shumen, Slavcho Nikolov & 

co, Shumen, pp. 199. 




flowering plants: APG II, The Linnean Society 

of London, Botanical Journal of the Linnean 

Society, 141: 399–436. 


2006. Conspectus of the Bulgarian vascular 

flora. Distribution maps and floristic elements, 

Bulgarian Biodiversity Foundation, Sofia, 452 

pp. 

[21] GRUEV, B., Kuzmanov B., 1994. General 




Management plan 2001, 2010. Adopted by 


04.07.2001, Sofia, 338 pp. 


















Balcanica 15 (1): 63–94. 


special arrangements for the conservation and use 

of medicinal plants, State Gazette number 16 


31


A CHARACTERISTIC OF MODEL HABITATS IN SOUTH DOBRUDJA 

Dimcho ZAHARIEV 


115 Universitetska Str., 9712, Shumen, Bulgaria, dimtchoz@yahoo.com 

__________________________________________________________________________________________ 

Abstract: Five natural habitats and five artificial habitats (forest shelter belts) are investigated in South 

Dobrudja. Most taxonomical diversity and most protected species from natural habitats are established in Western 

Pontic Paeonian steppes near to Bejanovo village. In the forest shelter belts is typical less taxonomical diversity, 

less protected species and more anthropophytes, which due to strong anthropogenically influence. The families 

with most of genera and species are: Asteraceae, Рoaceae, Rosaceae and Lamiaceae. The biggest groups from 

biological types are perennial herbaceous plants and annual herbaceous plants. The floristic elements are 

presented mainly from circumboreal, European and Mediterranean elements. The mainly reasons about high 

number of anthropophytes are intensive fragmentation of the natural habitats, all round from agricultural areas – a 

source of anthropophytes, and their accessibility for peoples and domestic animals. 

Keywords: Dobrudja, habitats, taxonomical diversity, biological types, floristic elements, endemites, relict 

species, protected species, anthropophytes. 

__________________________________________________________________________________________ 


Dobrudja is historical and geographical area 

between the lower reaches of the Danube and Black 

Sea. The area is 23 000 km 2 . It is divided into two 

parts – North and South Dobrudja. 

North Dobrudja is located in Southeastern 

Romania. Its area covers about 2/3 of the territory, 

amounting to 15 435 km². 

South Dobrudja is located in Northeastern 

Bulgaria. The area is 7 565 km². The Bulgarian part 

of Dobrudja is divided by the virtual line between 

Stojer village and Rosica village into two parts – 

eastern and western. South Dobrudja is located in 3 

administrative areas – Varnenska (municipality 

Aksakovo), Dobrichka (all municipalities) and 

Silistrenska (municipality Kainardja). 

The climate is temperate. It is characterized by 

warm summers and cold winters, high annual 

amplitude of air temperature, spring–summer 

minimum and winter maximum of rainfall, the snow 

cover is relatively stable. The average temperatures in 

January are between 0°С and –1.5°С. In the summer 

dominated tropical and subtropical air masses and the 

average temperature in July is 22-24°С. The spring 

and the autumn are approximately the same 

temperatures. April was warmer in October. The 

rainfalls are with maximum in May–June and with 

minimum in February–March. The annual amount of 

precipitation is 520 to 650 mm. About 10% of the 

total amount of precipitation is snow [1]. 

In South Dobrudja dominated haplic Chernozems. 

Small areas are covered with kastanic, calcaric 

or gleyc Chernozems. On the coast of the Black Sea 

and the rivers are distributed rendzic Leptosols and 

Nitisols. Along the Danube are distributed calcaric 

Fluvisols, Histosols and Gleysols. Unique to the 

region are the small in area Vertisols [2]. 

In terms of its flora, South Dobrudja belongs to 

the region of Northeastern Bulgaria. On its territory 

are described 1 508 species, which are referred to 496 

genera and 144 families. Natural vegetation was 

composed of forest steppes, which include large 

forest complexes and grasslands. Original natural 

vegetation of the Dobrudja was destroyed in a large 

part, due to intensive human activities [3]. Today on 

the territory of Southern Dobrudja occur 41 different 

plant communities – primary and secondary [4]. 33 

habitats are described according to Council Directive 

92/43/EEC of the European Community to protect 


A characteristic of model habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) 

natural habitats and of wild fauna and flora [5, 6, 7, 8, 

9, 10, 11, 12, 13, 14]. 


The field studies were conducted on the route 

method in 2008 – 2009. Subject of research are a 

total of 10 different habitats – 5 natural and 5 

artificial. The natural habitats are defined by 

Kavrakova et all. [15]. Each habitat is characterized 

as follows: average altitude, exposure, slope, area, 

soil type and subtype, base rock, cover of tree, shrub 

and herbaceous vegetation, number of established 

species, genera and families, cover of each species, 

distribution in biological type, floristic elements, 

endemites, subendemites and relict species, species 

with conservation status, anthropophytes, 

anthropogenic influence. The average altitude, 

exposure, slope and area are defined with map at a 

scale 1:50 000. The soil types and subtypes are 

presented by Ninov [2]. The taxons and the biological 

type are defined by Identifier of the vascular plants in 

Bulgatia [16], Flora of PR Bulgaria, Vol. І – Х [17]. 

The update of the taxons is consistent with APG II 

[18] and Petrova et all. [19]. The cover of each 

species is presented by Braun-Blanquet [20]. The 

following symbols are used: r – cover less than 5%, 

one individual; + – cover less than 5%, 2-5 

individuals; 1 – cover less than 5%, 6-50 individuals; 

2m – cover less than 5%, more than 50 individuals; 

2a – cover 5-12.5%; 2b – cover 12.5-25%; 3 – cover 

25-50%; 4 – cover 50-75%; 5 – cover 75-100%. The 

following symbols are used [16] for biological types: 

t (from English tree), sh (from English shrub), p 

(from English perennial), а (from English annual). 

The floristic elements, endemites and subendemites 

are presented by Asiov et all. [21]. The relicts are 

presented by Gruev and Kuzmanov [22], Peev [23], 

Boža et all. [24], Peev et all. [25]. The conservation 

status is presented using the following documents: 


Community to protect natural habitats and of wild 

fauna and flora [26], Berne Convention [27], 

Convention on International Trade 

in Endangered Species of Wild Fauna and Flora 

(CITES) [28], Red book of PR Bulgaria [29], IUCN 

Red List for Bulgaria [30], Biological Diversity Act 

[27], Order for special arrangements for the 

conservation and use of medicinal plants [31]. The 

34 

anthropophytes are presented by Stefanov and 

Kitanov [32]. is recorded by the system of effects 

used in the assessment of an object from the network 

of protected areas Nature 2000. 


HABITAT 1 

A habitat by Nature 2000: Euro-Siberian 

steppic woods with Quercus spp. 

A habitat by Bondev [4]: Cerris oak (Querceta 

cerris) forests. 

It is located southwest of Efreitor Bakalovo 

village, municipality Krushari. The territory is a part 

of Nature 2000 (Protected area “Suha reka”). The 

average altitude is 150 m. The exposure is south. The 

slope varies in different parts. It is smaller in the 

north and higher in southern parts. The area of the 

habitat is 6 300 dka. The soil type is Chernozems, 

and the soil subtype is haplic Chernozems. The 

bedrock is limestone. The cover of the tree vegetation 

is 80%, the cover of the shrub vegetation is 10% and 

the cover of the herbaceous vegetation is 10%. 

In the habitat have been indetified 78 species of 

vascular plants from 67 genera and 27 families. The 

families with greatest number of genera are as 

follows: Asteraceae – 8 (11.94%), Poaceae – 8 

(11.94%), Brassicaceae – 5 (7.46%) and Fabaceae – 

5 (7.46%). The families with greatest number of 

species are as follows: Poaceae – 10 (12.82%), 

Rosaceae – 9 (11.54%), Asteraceae – 8 (10.26%), 

Brassicaceae – 6 (7.69%), Fabaceae – 5 (6.41%), 

Lamiaceae – 5 (6.41%) and Scrophulariaceae – 5 

(6.41%). The genera with greatest number of species 

are as follows: Veronica – with 4 species (5.13%), 

Poa – with 3 species (3.85%) and Potentilla – with 3 

species (3.85%). 

With the highest percentage of coverage are 

Quercus cerris L. (5) and Poa nemoralis L. (2a). 

With the lowest percentage of coverage (1) are 

Cornus mas L., Carduus nutans L. and Vicia sativa 

L. Each of the remaining 73 species has coverage 2m. 

The distribution of species in biological type is 

as follows: The perennial herbaceous plants (p) are 

most – they are 38 species (48.72%). Secondly, are

Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010) 

annual herbaceous plants (a) with 21 species 

(26.92%). The next is the transition group of annual 

to biennial herbaceous plants (a-b) with 6 species 

(7.69%). The trees (t) and the transition group of 

shrubs to trees (sh-t) have equal number of species – 

4 (5.13%). The biennial herbaceous plants (b) are 3 

species (3.85%), and shrubs (sh) are 2 species 

(2.56%) only. 

The diversity of floristic elements is as follows: 

The largest number of species (28) has circumboreal 

origin. The next are species with European origin – 

they are 25 species. 13 species have Mediterranean 

origin. The Pontic type of elements and 

cosmopolitans are 5 species each of them. One of the 

species is adventive element. One of the species is 

Balkan subendemite – Ornithogalum sibthorpii 

Greut. 

Three species are Tertiary relicts: Carpinus 

orientalis Mill., Quercus cerris L. and Ulmus minor 

Mill. 

Two species with protection statute are 

established – Crocus flavus West. and Scilla bifolia 

L. They are included in the Biological Diversity Act 

in the category „Under the protection and regulated 

use of nature”. 

The anthropophytes are 55 species (70.51%). 

The large number indicates for increased 

anthropogenic impact on the habitat. 

The anthropogenic influence consists in the 

following: 1. Forestry felling. 2. Artificial 

afforestation. 3. Grazing sheep, goats and cows. 4. 

Pollution by garbage from the shepherds and farm 

workers. 5. Arable land in the vicinity. 6. Improved 

access to the habitat by a system of paths and roads. 

7. Тourist pavilion with a fireplace. 8. Fountain with 

several troughs. 

HABITAT 2 

A habitat by Bondev [4]: Shrub (Amygdaleta 

nanae) and grass (Artemisieta albae, Agropyreta 

pectiniformae, Agropyreta brandzae, Brometa riparii 

etc.) steppe and xerothermal communities. 

It is located south of Karapelit village, 

municipality Dobrich. The territory is a part of Nature 

2000 (Protected area “Suha reka”). The average 

altitude is 160 m. The exposure is in some parts 

south, while in others – west. The slope is variable 

35 

and reaches 30°. The area of the habitat is 400 dka. 

The soil type is Chernozems, and the soil subtype is 

haplic Chernozems. The bedrock is limestone. The 

cover of the shrub vegetation is less 

than 5% and the cover of the herbaceous vegetation is 

90%. More than 5% of the ground is devoid of 

vegetation cover. 




follows: Rosaceae – 10 (14.29%), Asteraceae – 7 

(10.00%), Lamiaceae – 7 (10.00%), Poaceae – 7 

(10.00%) and Apiaceae – 5 (7.14%). The families 

with greatest number of species are as follows: 

Rosaceae – 10 (12.05%), Lamiaceae – 10 (12.05%), 

Asteraceae – 9 (10.84%), Poaceae – 7 (8.43%), 

Apiaceae – 5 (6.02%), Caryophillaceae – 5 (6.02%) 

and Ranunculaceae – 5 (6.02%). The genera with 

greatest number of species are as follows: Euphorbia 

and Salvia – with 3 species each of them (3.61%). 

Stipa capillata L. (2а) is with the highest 

percentage of coverage. With the lowest percentage 

of coverage are Robinia pseudoacacia L. (1), Althaea 

cannabina L. (1), Prunus mahaleb L. (+), Carduus 

nutans L. (+), Pyrus pyraster Burgsd. (+), Ligustrum 

vulgare L. (r) and Malus sylvestris Mill. (r). From the 

neighboring shelter belt immigrated some tree 

species. The reason for this is the transference of 

fruits and seeds by birds and wind. Each of the 

remaining 75 species has coverage 2m. 



most – they are 48 species (57.83%). Secondly, are 


(21.69%). The biennial herbaceous plants (b) and the 

trees have 4 species each of them (4.82%). The 

shrubs (sh) are 3 species (3.61%). The transition 

group of shrubs to trees (sh-t) and the transition group 

of annual to perennial herbaceous plants (a-р) have 2 

species each of them (2.41%). The transition groups 

of annual to biennial herbaceous plants (a-b) and of 

biennial to perennial herbaceous plants (b-р) have 

one species (1.20%) each of them. 

The most species are species with Mediterranean 

(23 species), European (22 species) and circumboreal 

origin (20 species). The Pontic type of elements are 

10 species. The cosmopolitans are 3 species. The 

Balkan endemites are 2 species – Achillea clypeolata


Sibth. et Sm. and Salvia ringens Sibth. et Sm. One 

species is Balkan subendemite – Dianthus pallens 

Sm. One species has adventive origin and one species 

has Alpine-Mediterranean. 

Four species with protection statute are 

established: Adonis vernalis L. is included in CITES 

and in the Order for special arrangements for the 

conservation and use of medicinal plants in the 

category “Collecting herbs is prohibited from the 

natural habitats”. Jurinea ledebourii Bunge is 

included in the IUCN Red List for Bulgaria in the 

category “Endangered”, in the Red book for Bulgaria 

in the category „Rare” and in the Biological Diversity 

Act in the category „Protected”. Two species are 

included in the Biological Diversity Act in the 

category „Under the protection and regulated use of 

nature”: Bupleurum affine Sadl. and Stipa capillata 

L. 


They are an indicator of the extent of human impact 

on habitat. 

The anthropogenic influence on the habitat due 

to the presence of: 1. Improved access to the habitat 

by a system roads. 2. Arable land in the vicinity. 3. 

The forest shelter belts in the vicinity. 

HABITAT 3 

A habitat by Bondev [4]: Mesoxerothermal 

grass vegetation with a prevalence of Poa bulbosa L., 

Lolium perenne L., Cynodon dactylon L., partly also 

Dichantium ischaemum (L.) Roberty and rarely 

Chrysopogon gryllus (L.) Tryn. 

It is located between Izvorovo and Krasen 

villages, municipality General Toshevo. The average 

altitude is 180 m. The exposure is southwest. The 

slope is variable and reaches 30°. The area of the 

habitat is 2 000 dka. The soil type is Leptosols, and 

the soil subtype is rendzic Leptosols. The bedrock is 

limestone. The cover of the shrub vegetation is less 







follows: Asteraceae – 15 (14.56%), Poaceae – 12 

(14.29%), Lamiaceae – 10 (11.90%) and 

36 

Brassicaceae – 5 (5.95%). The families with greatest 

number of species are as follows: Poaceae – 14 

(13.59%), Lamiaceae – 13 (12.62%), Asteraceae – 12 

(11.65%), Brassicaceae – 5 (4.85%), Euphorbiaceae 

– 5 (4.85%) and Rubiaceae – 5 (4.85%). The genus 

Euphorbia is with greatest number of species – with 5 


With the highest percentage of coverage are Poa 

pratensis L. (2b) and Elymus repens (L.) Gould. (2а). 

With the lowest percentage of coverage (1) is 

Carduus thoermeri Weinm. Each of the remaining 

100 species has coverage 2m. 





(31.07%). The biennial herbaceous plants (b) are 10 

species (9.71%). The transition group of annual to 

biennial herbaceous plants (a-b) has 3 species 

(2.91%). The transition group of annual to perennial 

herbaceous plants (a-р) has 2 species (1.94%). The 

trees (t) are 2 species (1.94%) and the shrubs (sh) – 

one species (3.61%) only. 

The largest number of species (31) has 

circumboreal origin. Secondly, are European (27 

species) and Mediterranean type of elements (20 

species). The species with Pontic origin are 13. The 

cosmopolitans are 6 species. Three species are 

Balkan subendemites – Ornithogalum sibthorpii 

Greut., Verbascum banaticum Schrad. and Carduus 

thoermeri Weinm. The adventive species are 2. One 

species has Alpine-Carpathian origin. 

Three species with protection statute are 

established: Artemisia pedemontana Balb. is included 

in IUCN Red List for Bulgaria in the category 

„Endangered”, in the Red book for Bulgaria in the 

category „Threatened with extinction” and in the 

Biological Diversity Act in the category „Protected”. 

Helichrysum arenarium (L.) Mornh. and Stipa 

capillata L. are included in the Biological Diversity 

Act in the category “Under the protection and 

regulated use of nature”. Helichrysum arenarium (L.) 

Mornh. is included in the Order for special 


medicinal plants in the category “Collecting herbs is 

prohibited from the natural habitats”.



They show a significant anthropogenic impact on the 

habitat. 




Transmission line, passing through the territory. 4. 

Grazing sheep and goats. 5. Pollution by garbage 

from the two villages. 6. Artificial terracing of slopes. 

HABITAT 4 

A habitat by Nature 2000: Western Pontic 

Paeonian steppes 

It is located near Bejanovo village, municipality 

General Toshevo. The territory is a part of Nature 

2000 (Protected area “Kraimorska Dobrudja”). The 

average altitude is 80 m. The exposure is northeast. 

The slope is low and reaches 5°. The area of the 

habitat is 650 dka. The soil type is Chernozems, and 

the soil subtype is calcaric Chernozems. The bedrock 

is limestone. The cover of the shrub vegetation is less 





vascular plants from 116 genera and 36 families. It is 

the richest of plant species from the natural habitats. 

The families with greatest number of genera are as 

follows: Asteraceae – 13 (11.21%), Rosaceae – 12 

(10.34%), Lamiaceae – 11 (9.48%), Poaceae – 11 

(9.48%), Boraginaceae – 6 (5.17%), Brassicaceae – 6 

(5.17%), Fabaceae – 6 (5.17%), Apiaceae – 5 

(4.31%), Ranunculaceae – 5 (4.31%) and 

Scrophulariaceae – 5 (4.31%). The families with 

greatest number of species are as follows: Asteraceae 

– 18 (11.76%), Rosaceae – 17 (11.11%), Lamiaceae 

– 15 (9.80%), Poaceae – 14 (9.15%), Boraginaceae – 

8 (5.23%), Caryophyllaceae – 8 (5.23%), Fabaceae – 

7 (4.58%), Apiaceae – 6 (3.92%), Brassicaceae – 6 

(3.92%), Ranunculaceae – 6 (3.92%), Euphorbiaceae 

– 5 (3.27%), Rubiaceae – 5 (3.27%) and 

Scrophulariaceae – 5 (3.27%). The genera with 

greatest number of species are as follows: Euphorbia 

– with 5 species (3.27%), Cerastium, Potentilla, 

Prunus, Salvia and Silene – with 3 species each of 

them (1.96%). 

With the highest percentage of coverage (2b) are 

Festuca pseudovina Hack. ex Wiesd., Poa pratensis 

37 

L. and Stipa capillata L. With the cover 1 are 14 

species. With the lowest percentage of coverage are 

Malus dasyphylla Borkh. (+) and Cydonia oblonga 

Mill. (r). They are most likely carried by birds. Each 

of the remaining 134 species has coverage 2m. 





(24.84%). The next is the transition group of annual 

to biennial herbaceous plants (a-b) with 7 species 

(4.58%). The trees (t) and the shrubs (sh) are 5 

species each of them (3.27%). The biennial 

herbaceous plants (b) are 4 species (2.61%). The 

transition groups of annual to perennial herbaceous 

plants (a-р), of biennial to perennial herbaceous 

plants (b-p) and of shrubs to trees (sh-t) have 2 

species each of them (1.31%). 


circumboreal origin. The next are species with 

Mediterranean and European type of elements – with 

37 species each of them. Thirdly, are the Pontic type 

of elements with 21 species. The cosmopolitan are 6 

species. Four of the species are Balkan endemites – 

Achillea clypeolata Sibth. et Sm., Astragalus 

spruneri Boiss., Chamaecytisus jankae (Vel.) Rothm. 

and Potentilla emili-popii Nyar. Five of the species 

are Balkan subendemites – Carduus thoermeri 

Weinm., Centaurea napulifera Roch., Dianthus 

pallens Sm., Ornithogalum sibthorpii Greut. and 

Thesium simplex Vel. The remaining 3 species have 

Alpine-Mediterranean, Oriental-Turanian and 

Pannonian-Pontic origin. 

Eight species with protection statute are 

established: Paeonia tenuifolia L. is included in 

Berne Convention, in Directive 92/43/ЕЕС and in the 

Biological Diversity Act in the category “Protected”. 

Potentilla emili-popii Nyar. is included in Berne 

Convention, in Directive 92/43/ЕЕС, in the 

Biological Diversity Act in the category „Declaration 

of protected areas to protect habitat for species by 

Directive 92/43/ЕEC” and in the category 

„Protected”. Adonis vernalis L. is included in CITES 




natural habitats”. Artemisia pedemontana Balb. is 

included in IUCN Red List for Bulgaria in the


category „Endangered”, in the Red book for Bulgaria 

in the category „Threatened with extinction” and in 

the Biological Diversity Act in the category 

„Protected”. Erodium hoefftianum C. A. Mey. is 

included in the Red book for Bulgaria in the category 

„Rare” and in IUCN Red List for Bulgaria in the 

category „Near Threatened”. Pulsatilla montana 

(Hoppe) Reichenb., Stipa capillata L. and Stipa 

lessingiana Trin. et Rupr. are included in the 

Biological Diversity Act in the category “Under the 

protection and regulated use of nature”. 

The anthropophytes are 92 species (60.13%), 

which indicates a high anthropogenic impact on the 

habitat. 




The forest shelter belts and artificial forest from 

Robinia pseudoacacia L. in the vicinity. 4. Grazing 

cows. 5. Disposal of soil in the vicinity. 

HABITAT 5 

A habitat by Nature 2000: Rupicolous 

calcareous or basophilic grasslands of the Alysso- 

Sedion albi. 

It is located between Onogur and Efreitor 

Bakalovo villages, municipality Krushari. The 

territory is a part of Nature 2000 (Protected area 

“Suha reka”). The average altitude is 70 m. The 

exposure is south. The slope is variable and reaches 

40°. The area of the habitat is 30 dka. The soil type is 

Leptosols, and the soil subtype is rendzic Leptosols. 

The bedrock is limestone. The cover of the shrub 

vegetation is less than 5% and the cover of the 

herbaceous vegetation is 30%. More than 65% of the 

ground is devoid of vegetation cover. 


vascular plants from 43 genera and 22 families. It is 

the most poor of plant species from the natural 

habitats. The families with greatest number of genera 

are as follows: Asteraceae – 7 (16.28%), Рoaceae – 5 

(11.63%), Lamiaceae and Apiaceae – with 4 species 

each of them (9.30%). The families with greatest 

number of species are as follows: Asteraceae – 8 

(17.39%), Lamiaceae and Poaceae – with 5 species 

each of them (10.87%). The genera with greatest 

number of species are as follows: Centaurea, Sedum 

and Teucrium – with 2 species each of them (4.35%). 

38 

With the highest percentage of coverage (2а) is 

Dichantium ischaemum (L.) Roberty. With the lowest 

percentage of coverage (+) are Gleditsia triacanthos 

L., Cornus sanguinea L. and Prunus spinosa L. Each 

of the remaining 42 species has coverage 2m. 

The perennial herbaceous plants (p) are most – 

they are 24 species (52.17%). Secondly, are annual 

herbaceous plants (a) with 8 species (17.39%). The 

shrubs are 6 species (13.04%). The biennial 

herbaceous plants (b) are 3 species (6.52%). The 

transition group of shrubs to trees (sh-t) has 2 species 

(4.35%). The trees (t), the transition groups of annual 

to biennial herbaceous plants (а-b) and of annual to 

perennial herbaceous plants (a-р) have one species 

(2.17%) each of them. 


Mediterranean origin. Secondly, are circumboreal 

type of elements with 11 species. The next are species 

with Pontic (9 species) and European origin (8 

species). The cosmopolitan are 3 species. One of the 

species is adventive element. 

Two species with protection statute are 

established: Stipa capillata L. is included in the 

Biological Diversity Act in the category „Under the 

protection and regulated use of nature”. Sedum acre 

L. is included in the Order for special arrangements 

for the conservation and use of medicinal plants in 

the category “Under a restrictive regime”. 


The high rate is due to human activities in adjacent 

areas of the habitat. 



by a system roads. 2. Grazing cows in the bottom of 

the slope. 3. Arable land in the vicinity. 

HABITAT 6 

Forest shelter belt formed by Quercus cerris L. 

It is located between General Toshevo and 

Liuliakovo village, municipality General Toshevo. 

The average altitude is 210 m. The exposure is west. 

The shelter belt is oriented in a southwest – northeast 

direction. The slope is low and reaches 5°. The area 

is 75 dka. The length of the shelter belt is 5 000 m, 

and the width – 15 m. The soil type is Chernozems, 

and the soil subtype is haplic Chernozems. The 

bedrock is limestone. The cover of the tree vegetation


is 80%, the cover of the shrub vegetation is 10% and 

the cover of the herbaceous vegetation is 60%. 

In the shelter belt have been indetified 49 species 

of vascular plants from 44 genera and 21 families. 


follows: Asteraceae and Рoaceae – with 8 species 

each of them (18.18%), Rosaceae – 6 (13.64%) and 

Lamiaceae – 4 (9.09%). The families with greatest 

number of species are as follows: Asteraceae and 

Рoaceae – with 8 species each of them (16.33%), 

Rosaceae – 6 (12.24%) and Lamiaceae – 4 (8.16%). 

The genera with greatest number of species are as 

follows: Avenula, Cirsium, Galium, Prunus and 

Sambucus – with 2 species each of them (4.08%). 

With the highest percentage of coverage (5) are 

Quercus cerris L. and Poa pratensis L. (3). With 

coverage 2b are Avenula compressa (Heuff.) Sauer et 

Chmelit., Avenula pubescens (Huds.) Dumort. and 

Lolium perenne L. With coverage 2a are Robinia 

pseudoacacia L. and Hordeum hystrix Roth. With the 

lowest percentage of coverage (+) are Crataegus 

monogyna Jacq., Cirsium arvense (L.) Scop. and 

Euphorbia agraria Bieb. Only with one individual (r) 

is Celtis australis L. Each of the remaining 38 species 

has coverage 2m. 



herbaceous plants (a) with 10 species (20.41%). 

Thirdly, are the shrubs (sh) with 5 species (10.20%). 

The trees (t) are 4 species (8.16%). The transition 

group of shrubs to trees (sh-t) has 3 species (6.12%). 

The biennial herbaceous plants (b) are 2 species 

(4.08%). The transition group of annual to biennial 

herbaceous plants (а-b) has 1 species (2.04%) only. 


circumboreal origin. Secondly, are species with 

European (11) and Mediterranean origin (10). The 

cosmopolitan are 3 species. Two of the species are 

adventive elements. One of the species has Pontic 

origin. One of the species is Balkan subendemite – 

Galium pseudoaristatum Schur. 

One species is Tertiary relict in all of the habitat 

– Quercus cerris L. It is wooded artificial for the 

creation of the shelter belt. 

There are no species of protection status. This 

can be explained easily by the artificial origin of the 

habitat. 

39 


The high number is due to the artificial origin of the 

habitat and adjacent to farmland. 

The anthropogenic influence due to the presence 

of: 1. Improved access to the habitat by a system 

roads. 2. Grazing goats and cows. 3. Pollution by 

garbage from the shepherds and farm workers. 4. 

Arable land in the vicinity. 

L. 

HABITAT 7 

Forest shelter belt formed by Fraxinus excelsior 

It is located near Chernookovo village, 

municipality General Toshevo. The average altitude 

is 160 m. The exposure is east. The shelter belt is 

oriented in a southwest – northeast direction. The 

slope is low and reaches 5°. The area is 31.5 dka. The 

length of the shelter belt is 2 100 m, the width – 15 

m, and the height – 15 m. The soil type is 

Chernozems, and the soil subtype is haplic 

Chernozems. The bedrock is limestone. The cover of 

the tree vegetation is 80%, the cover of the shrub 

vegetation is 10% and the cover of the herbaceous 

vegetation is 60%. 




follows: Asteraceae – 11 (22.00%), Рosaceae – 6 

(12.00%) and Rosaceae – 6 (12.00%). The families 


Asteraceae – 14 (23.73%), Рosaceae – 8 (13.56%) 

and Rosaceae – 6 (10.17%). The genera with greatest 

number of species are as follows: Chenopodium and 

Fraxinus – with 3 species each of them (5.08%); 

Artemisia, Bromus, Carduus, Centaurea, Consolida 

and Hordeum – with 2 species each of them (3.39%). 

With the highest percentage of coverage (5) is 

Fraxinus excelsior L., followed by Poa pratensis L. 

(2а). With the lowest percentage of coverage (+) are 

Amorpha fruticosa L. and Elaeagnus angustifolia L. 

Only with one individual (r) is Salvia argentea L. 

With the cover 1 are 4 species. Each of the remaining 

50 species has coverage 2m. 




Thirdly, are the trees (t) with 7 species (11.86%). The


shrubs (sh), the biennial herbaceous plants (b) and the 

transition group of annual to biennial herbaceous 

plants (а-b) have 4 species each of them (6.78%). The 

transition group of shrubs to trees (sh-t) has 2 species 

(3.39%). 



European (13) and Mediterranean origin (8). The 

cosmopolitan are 6 species. Five of the species have 

Pontic origin. Four of the species are adventive 

elements. 

Four species in the habitat are Tertiary relicts: 

Acer tataricum L., Cotinus coggygria Scop., 

Fraxinus excelsior L., Quercus cerris L. The main 

species is Fraxinus excelsior L. It is wooded artificial 

for the creation of the shelter belt. Acer tataricum L. 

and Cotinus coggygria Scop. have less than 5% 

coverage and their number is more than 50 

individuals. The number of the individuals from 

Quercus cerris L. is less than 50. Perhaps individuals 

of these three species have evolved from fruit, carried 

over from adjacent areas. 

One species with protection statute is established 

– Fraxinus pallisiae Wilmott. It is included in IUCN 

Red List for Bulgaria in the category „Vulnerable”. It 

has less than 5% coverage, and its number is more 

than 50 individuals. The most likely reason for its 

presence in the shelter belt is its planting together 

with basic species Fraxinus excelsior L. 


Extremely high number of them due to the artificial 

origin of the habitat and adjacent to farmland. 



roads. 2. Pollution by garbage from the shepherds and 

farm workers. 3. Arable land in the vicinity. 

HABITAT 8 

Forest shelter belt formed by Fraxinus oxycarpa 

Willd. 

It is located as a third shelter belt between 

Vladimirovo and Benkovski villages, municipality 

Dobrich. The average altitude is 230 m. The exposure 

is northeast. The shelter belt is oriented in a 

southwest – northeast direction. The slope is low and 

reaches 5°. The area is 17 dka. The length of the 

shelter belt is 1 150 m, the width – 15 m, and the 

height – 15 m. The soil type is Vertisols, and the soil 

40 

subtype is eutric Vertisols. The bedrock is limestone. 

The cover of the tree vegetation is 80%, the cover of 

the shrub vegetation is 10% and the cover of the 

herbaceous vegetation is 60%. 




follows: Asteraceae – 9 (19.15%), Rosaceae – 8 

(17.02%) and Рosaceae – 6 (12.77%). The families 


Asteraceae – 10 (18.18%), Rosaceae – 10 (18.18%) 

and Рosaceae – 7 (12.73%). The genera with greatest 

number of species are as follows: Acer and Prunus – 

with 3 species each of them (5.45%). 


Fraxinus oxycarpa Willd. With the lowest percentage 

of coverage (1) are Amorpha fruticosa L., Tilia 

cordata Mill. and Tilia tomentosa Moench. Each of 

the remaining 51 species has coverage 2m. 




Thirdly, are the trees (t) with 9 species (16.36%). The 

shrubs (sh) and the transition group of shrubs to trees 

(sh-t) have 4 species each of them (7.27%). The next 

is the transition group of annual to biennial 

herbaceous plants (a-b) with 2 species (3.64%). The 

biennial herbaceous plants (b), the transition groups 

of annual to perennial herbaceous plants (а-р) and of 

biennual to perennial herbaceous plants (b-р) are 

presented with one species (1.82%) only. 

The largest number of species (25) has circumboreal 

origin. Secondly, are species with European 

(13) and Mediterranean origin (7). The cosmopolitan 

are 4 species. Three of the species have Pontic origin. 

Three of the species are adventive elements. 

Three species in the habitat are Tertiary relicts: 

Acer campestre L., Acer tataricum L., Quercus cerris 

L. Each of them has less than 5% coverage and their 

number is more than 50 individuals. The reason for 

their presence can be traced in the transference of 

fruit from neighboring areas. 

There are no species of protection status. This 

can be explained easily by the artificial origin of the 

habitat.







roads. 2. Pollution by garbage. 3. Arable land in the 

vicinity. 

HABITAT 9 

Forest shelter belt formed by Gleditsia 

triacanthos L. 

It is located south of Karapelit village, 

municipality Dobrich. The average altitude is 175 m. 

The exposure is northeast. The shelter belt is oriented 

in a north – south direction. The slope is low and 

reaches 10°. The area is 15 dka. The length of the 

shelter belt is 1 000 m, the width – 15 m, and the 

height – 15 m. The soil type is Chernozems, and the 

soil subtype is haplic Chernozems. The bedrock is 

limestone. The cover of the tree vegetation is 60%, 

the cover of the shrub vegetation is 10% and the 

cover of the herbaceous vegetation is 70%. 


of vascular plants from 78 genera and 30 families. It 

is the richest of plant species from the shelter belts. 

The reason for this is that is located immediately 

adjacent steppe. From the steppe to the shelter belt 

migrated a large number of species. The families with 

greatest number of genera are as follows: Lamiaceae 

– 11 (14.10%), Fabaceae – 9 (11.54%), Rosaceae – 8 

(10.26%), Asteraceae – 7 (8.97%), Poaceae – 6 

(7.69%) and Apiaceae – 5 (6.41%). The families with 

greatest number of species are as follows: Lamiaceae 

– 13 (14.77%), Rosaceae – 11 (12.50%), Asteraceae 

– 9 (10.23%), Fabaceae – 9 (10.23%), Poaceae – 6 

(6.82%) and Apiaceae – 5 (5.68%). The genus with 

greatest number of species is Prunus – with 4 species 

(4.55%). 


Gleditsia triacanthos L. Secondly, it is Poa pratensis 

L. (2а). With the cover 1 are 23 species. With the 

lowest percentage of coverage are Carduus 

acanthoides L., Sanguisorba minor Scop. (+) and 

Verbascum ovalifolium Sms. (r). Each of the 

remaining 60 species has coverage 2m. 




41 

Thirdly, are the trees (t) and the shrubs (sh) with 9 

species (10.23%) each of them. The next are the 

transition group of shrubs to trees (sh-t) and the 

biennial herbaceous plants (b) with 4 species each of 

them (4.55%). The transition group of annual to 

perennial herbaceous plants (а-р) has 2 species 

(2.27%). The transition groups of annual to biennial 

herbaceous plants (а-b) and of biennual to perennial 

herbaceous plants (b-р) are presented with one 




Mediterranean origin (21). The next are species with 

European (14) and Pontic origin (12). Six of the 

species is adventive element. The cosmopolitan are 5 

species. One of the species has Oriental-Turanian 

origin. One of the species is Balkan endemite – 

Achillea clypeolata Sibth. et Sm. 

Three species are Tertiary relicts: Celtis australis 

L., Cotinus coggygria Scop., Ulmus minor Mill. Each 

of them has less than 5% coverage. The number of 

Celtis australis L. is less than 50 individuals. The 

number of another two species is more than 50 

individuals. The reason for their presence can be 

traced in the transference of fruit from neighboring 

areas. 

Four species with protection statute are 

established: Adonis vernalis L. is included in CITES 




natural habitats”. Jurinea ledebourii Bunge is 

included in the IUCN Red List for Bulgaria in the 

category “Endangered”, in the Red book for Bulgaria 

in the category „rare” and in the Biological Diversity 

Act in the category „protected”. Tilia rubra DC. is 

included in IUCN Red List for Bulgaria in the 

category „Least Concern” and in the Red book for 

Bulgaria in category “Rare”. Asparagus officinalis L. 

is included in the the Biological Diversity Act in the 

category „Under the protection and regulated use of 

nature”. 

The presence of Adonis vernalis L. and Jurinea 

ledebourii Bunge is associated with their migration 

from the nearby steppe region.







roads. 2. Pollution by garbage from the shepherds and 

farm workers. 3. Arable land in the vicinity. 

HABITAT 10 

Forest shelter belt formed by Robinia 

pseudoacacia L. 

It is located as a first shelter belt east of the 

highway between Durankulak village and Durankulak 

Checkpoint, municipality Shabla. The average 

altitude is 20 m. The first half of the shelter belt is 

oriented in a west – east direction. The second half of 

the shelter belt is oriented in a northwest – southeast 

direction. The territory has no inclination. The area is 

15 dka. The length of the shelter belt is 1 000 m, the 

width – 15 m, and the height – 7 m. The soil type is 

Leptosols, and the soil subtype is rendzic Leptosols. 

The bedrock is limestone. The cover of 

the tree vegetation is 60%, the cover of the shrub 

vegetation is 10% and the cover of the herbaceous 

vegetation is 80%. 


of vascular plants from 40 genera and 19 families. It 

is the most poor of plant species from the shelter 

belts. The families with greatest number of genera are 

as follows: Asteraceae – 9 (22.50%) and Рosaceae – 

5 (12.50%). The families with greatest number of 

species are as follows: Asteraceae – 10 (20.83%), 

Рosaceae – 7 (14.58%) and Rosaceae – 6 (12.50%). 

The genera with greatest number of species are as 

follows: Prunus – 3 (6.25%), Artemisia, Elymus, 

Euphorbia, Fraxinus, Galium, Lamium and Poa – 

with 2 species each of them (4.17%). 


Robinia pseudoacacia L., followed by species 

Elymus repens (L.) Gould. and Elymus hispidus 

(Opiz) Meld. (3) and Poa pratensis L. (2а). With the 

lowest percentage of coverage (1) are 16 species. 

Each of the remaining 28 species has coverage 2m. 




Thirdly, are the trees (t) with 6 species (12.50%). The 

next are the shrubs (sh) and the transition group of 

42 

shrubs to trees (sh-t) with 4 species each of them 

(8.33%). The transition group of annual to biennial 

herbaceous plants (а-b) has 3 species (6.25%). The 

biennial herbaceous plants (b) are 2 species (4.17%). 

The transition group of biennual to perennial 

herbaceous plants (b-р) has one species (2.08%) only. 



Mediterranean origin (10). The next are species with 

european origin – they are 7 species. The 

cosmopolitan are 4 species. Three species have 

Pontic origin. Three of the species are adventive 

elements. One of the species has Oriental-Turanian 

origin. One of the species is Balkan subendemite – 

Carduus candicans Waldst. et Kit. 

In the habitat is meeting once Tertiary relict – 

Fraxinus excelsior L. Its coverage is less than 5%. 

The number of individuals is in the range 6 – 50. The 

reason for its presence can be traced in the 

transference of fruit from neighboring areas. 

One species with protection statute is established 

– Artemisia pedemontana Balb. It is included in 

IUCN Red List for Bulgaria in the category 

„Endangered”, in the Red book for Bulgaria in the 

category „Threatened with extinction” and in the 

Biological Diversity Act in the category „Protected”. 

Its presence can be explained by the transfer of the 

fruits by wind and finding favorable conditions, 

associated with good light in the shelter belt. 


Extremely high number of them due to the artificial 

origin of the habitat and adjacent to farmland. 



roads. 2. Arable land in the vicinity. 


From natural habitats are established most 

taxonomical diversity in Western Pontic Paeonian 

steppes near to Bejanovo village, and least 

taxonomical diversity in Rupicolous calcareous or 

basophilic grasslands of the Alysso-Sedion albi 

between Onogur and Efreitor Bakalovo villages. 

From forest shelter belts are established most 

taxonomical diversity in Forest shelter belt formed by 

Gleditsia triacanthos L., and least taxonomical 

diversity in Forest shelter belt formed by Robinia


pseudoacacia L. between Durankulak village and 

Durankulak Checkpoint. 

The families with greatest number of genera and 

species are as follows: Asteraceae, Рoaceae, 

Rosaceae and Lamiaceae. 

In the analysis of the biological types was 

established pattern common to all habitats: most 

numerous are perennial and annual herbaceous plants. 

This confirms the results of research of Kozuharov et 

all. [33]. 

In all habitats are most floristic elements with 

circumboreal, European and Mediterranean origin. 

The Tertiary relicts, which are established, are 

trees and one shrub. They often have a secondary 

origin for the habitats. 

The species with protection statute in natural 

habitats are most in Western Pontic Paeonian steppes 

near to Bejanovo village – they are 8 species. In the 

other habitats that number varies from 2 to 4 species. 

In the forest shelter belts can be found a small 

number of species with protection statute. Most often 

they have gone from adjacent areas. 

The number of anthropophytes in the natural 

habitats is a significant – from 60.13% to 77.67%. 

The reasons about this are mainly the following: the 

strong fragmentation of natural habitats, arable land 

in the vicinity – like source of anthropophytes, 

improved access to the habitats and their accessibility 

for people and domestic animals. In the forest shelter 

belts the anthropophytes quite naturally are more – 

from 73.86% to 87.50%. 

5. References 

[1] VELEV, S., 2002. Climatic zoning, in Kopralev, 

I. (main ed.). Geography of Bulgaria. Physical 

and socio-economic geography, Institute of 

Geography, BAS, Farkom, Sofia, 760 pp. 

[2] NINOV, N., 2002. Soils, in Kopralev, I. (main 

ed.). Geography of Bulgaria. Physical and socioeconomic 

geography, Institute of Geography, 

BAS, Farkom, Sofia, 760 pp. 

[3] KITANOV, B., Penev, I., 1980. Flora of 

Dobrudja, Nauka i Izkustvo, Sofia, 630 pp. 

[4] BONDEV, I., 1991. The vegetation of Bulgaria. 

Map in М 1:600 000 with explanatory text, 

University Press St. Kliment Ohridski, Sofia, 183 

pp. 

43 

[5] NATURA 2000 Standard Data Form for 

Protected Area „The Valley of Batova River” 

(BG0000102), Ministry of Environment and 

Waters of Bulgaria, 15 pp. 


Protected Area „Kraimorska Dobrudja” 




Protected Area „Durankulak Lake” 




Protected Area „Shabla – Ezeretz Lake” 




Protected Area „Suha reka” (BG0002048), 

Ministry of Environment and Waters of Bulgaria, 

13 pp. 


Protected Area „Kardam” (BG0000569), Ministry 

of Environment and Waters of Bulgaria, 10. 


Protected Area „Izvorovo – Kraishte” 

(BG0000570), Ministry of Environment and Waters 

of Bulgaria, 10 pp. 


Protected Area „Rositza – Loznitza” 




Protected Area „Complex „Kaliakra” 



[14] TZONEV, R., Rusakova, V., Dimitrov, М. 

Dimova, D., Belev, T. Kavrakova, V., 2004. 

Proposals for habitats for inclusion to Annex I on 



wild fauna and flora and Interpretation handbook 

of habitats in the European Union EUR 15/2, 

Report, World Wildlife Fund, Danube – 

Carpathian Program (WWF, DCP). 

[15] KAVRAKOVA, V., Dimova, D., Dimitrov, М., 

Tzonev, R., Belev, T. (editors), 2005. A 

guidance for identifying the habitats of European 

importance in Bulgaria, Geosoft, Sofia, 128.


[16] KOZUHAROV, S. (ed.), 1992. Identifier of the 

vascular plants in Bulgatia, Nauka i izkustvo, 

Sofia, 788 pp. 






flowering plants: APG II, The Linnean Society of 

London, Botanical Journal of the Linnean 

Society, 141: 399–436. 

[19] PETROVA, А., Anchev, М. Palamarev, Е., 

1999. How to recognize the plants in our nature. 

Char identifier. Prosveta, Sofia, 837 pp. 

[20] WESTHOFF, V., Maarel, E., 1973. The Braun- 

Blanquet Approach in: Tuxen, R. (Ed.), 

Handbook of vegetation science. Dr. W. Junk b. 

v. Publishers the Hague, p. 619-704. 


2006. Conspectus of the Bulgarian vascular flora. 

Distribution maps and floristic elements, 

Bulgarian Biodiversity Foundation, Sofia, 452 p. 

[22] GRUEV, B., Kuzmanov B., 1994. General 




Management plan 2001 – 2010. Adopted by 


04.07.2001, Sofia, 338 pp. 

















77 from 9 august 2002, pp. 9–42. Amended in 

State Gazette number 94 from 16.11.2007. 

44 








Balcanica 15 (1): 63–94. 


special arrangements for the conservation and 

use of medicinal plants, State Gazette number 16 


[32] STEFANOV, B., Kitanov B., 1962. Kultigenen 

plants and kultigenen vegetation in Bulgaria, 


[33] KOZUHAROV, S., Dimitrov, D., Lazarova, М., 

Kozuharova, Е., 1997. A characteristic of the 

flora and the vegetation of the natural plant 

complexes in Southern Dobrudja, Conference 

proceedings „Dobrudja and Kaliakra”, Academic 

publishing of Higher Agricultural Institute, 

Plovdiv, p. 42-58.


FLORISTIC ASPECTS OF THE HILLS OF CAMENA VILLAGE 

(TULCEA COUNTY) 

Marius FĂGĂRAŞ 

Ovidius University of Constanţa, Faculty of Natural and Agricultural Sciences, Department of Biology and 

Ecology, Mamaia Blvd, No. 124, 900527, Constanţa, Romania, fagarasm@yahoo.com 

__________________________________________________________________________________________ 

Abstract: This paper presents the flora on the hills in the vicinity of Camena locality. These hills have volcanic 

origin and are located in the south-east of the Babadag plateau. The hilly landscape with spectacular rock 

formations, the substrate made up of acidic volcanites and the climate specific to the forest steppe are the main 

factors that determined the variety of the vegetation made up of steppe meadows, rock formations, forests and 

bushes. The area is characterized by the presence of a considerable number of floral rarities, some endemic, 

other rare, vulnerable or endangered at national level. Despite all these, the flora of the area is still little known 

as there are no specialized papers. The enumeration of the vascular flora is accompanied by an analysis of the 

biological forms, of the floral elements, of the ecological categories, but also of the floral rarities present on 

these hills. 

Keywords: Camena hills, flora, life forms, floristic elements, ecological categories, rare and threatened flora. 

___________________________________________________________________________ 


The hills of Camena are located south of 

Ciucurovei Hills, in the south-east of the Babadag 

Plateau, in the vicinity of Camena village (Tulcea 

County). They are volcanic hills (Fig. 7), with a 

maximum altitude of approx. 190 meters, located at 

the southern end of the Peceneaga-Camena crevice 

which separates the Northern Dobrogea Plateau 

from the Central Dobrogea Plateau. The Hills of 

Camena look like a wide saddle framed towards the 

north-west and south-east by the hydrographic 

basins of two valleys: Camena valley and Ciamurlia 

valley. In the southern part of these hills is the 

Altan Tepe copper pyrite mine. 

The geological layer is made up of rhyolites 

(quartz porphyry) of Paleozoic age, volcanic rocks 

(acid volcanites) colored in pink-red, reddish-brown 

and violet. In the plane zone and on the eroded 

inclines, the rhyolites emerge on the surface as 

spectacular rock formations. Towards the base of 

the hills, the rocks are covered by a layer of loess 

(3-4 meters thick). The soils are represented by 

chernozem and lithosoils, the latter being present 

especially in the rocky zones. 

The climate is temperate-continental, with 

average annual temperatures of 10.5-11 0 C, while 

the average annual precipitations range between 

450 and 500 mm/year. As vegetation type, the Hills 

of Camena fit within the forest steppe zone. The 

vegetation is made up of steppe meadows, rock 

vegetation (on the plateaus), thermophile forests 

and bushes. 

The Hills of Camena represent an area of the 

Babadag Plateau which is interesting from the 

geological, landscape and botanical point of view, 

firstly because of the volcanic origin of the hills, of 

the rhyolites disposed as spectacular rock 

formations and of the floral rarities which can be 

encountered in this area. Despite these, the flora of 

these hills is little known and limited to the 

quotation of species in older specialized literature 

[1, 2, 3, 4, 5]. 


The field researches have been done between 

years 2008-2010, during the entire vegetation 

season in order to cover all the phenology stages. 

The plant taxa nomenclature follows the Flora 

ilustrată a României. Pteridophyta et 


Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) 

Spermatophyta [6], Flora Europaea [7, 8] and 

Flora României [4]. The life forms, floristic 

elements and ecological categories have been 

established on the base of the synthesis works 

Conspectul florei cormofitelor spontane din 

România [9] and Flora ilustrată a României. 

Pteridophyta et Spermatophyta [6]. The rare and 

threatened plant species was done according to the 

Romanian Red List [10] and the Romanian Red 

Book of the vascular plants [11]. 


The floristic researches carried out on the hills 

of Camena village have lead to identification of 178 

vascular taxa (168 species and 10 subspecies) 

(Table 1). Taxa found in the studied area belong to 

46 families and 38 classes of Spermatophyta 

Divisio. The following families are well 

represented as number of taxa (Fig. 1): Asteraceae 

(12,35%), Lamiaceae (10,67%), Poaceae (9,55%), 

Rosaceae si Brassicaceae (5,05%), Liliaceae, 

Caryophyllaceae si Fabaceae (cate 4,49%), 

Apiaceae (3,93%), Boraginaceae (3,37%), 

Ranunculaceae (2,80), Geraniaceae (2,24) and 

Scrophulariaceae (1,68%). 

14 

12 

10 

8 

% 

6 

4 

2 

0 

AST LAM POA ROS BRAS LIL CARY 

families 

FAB API BOR RAN GER SCR 

Fig. 1. Most important botanical families as the 

number of species (AST-Asteraceae; POA- 

Poaceae; LAM-Lamiaceae, ROS-Rosaceae; LIL- 

Liliaceae;CARY-Caryophyllaceae; FAB-Fabaceae; 

API-Apiaceae; BRAS-Brassicaceae; 

RAN-Ranunculaceae; BOR-Boraginaceae; 

GER-Geraniaceae; SCR-Scrophulariaceae) 

From the point of view of the biological forms 

(Fig.2), the dominant ones are the 

46 

hemicryptophytes (42.13%) and the annual and 

biennial terophytes (35.95%), present especially in 

the steppe meadows. Poorly represented are the 

phanerophytes (9.55%), which are included in 

forests (with Quercus petraea subsp. dalechampii, 

Quercus pubescens, Carpinus orientalis, Fraxinus 

ornus, Prunus mahaleb, Tilia tomentosa) and 

bushes (with Crataegus monogyna, Prunus spinosa, 

Cotinus coggygria, Ligustrum vulgarae, Rosa 

canina, Cornus mas) in the investigated area. 

The category of phanerophytes also includes 

alien species encountered in these hills, some of 

them invasive or potentially invasive (Robinia 

pseudacacia, Ailanthus altissima, Elaeagnus 

angustifolia). The geophytes (7.30%) and the 

camephytes (5.05%) are perennial species found 

especially in the grassy blanket from forests or 

forest edges. 

PH 

9,55% 

TH 

35,95% 

G 

7,30% 

CH 

5,05% 

H 

42,13% 

Fig. 2. The spectrum of biological forms 

(H-hemicriptofite; TH-therofite; PH-fanerofite; 

G-geofite; CH-camefite) 

Among the floristic elements (Table 2 and 

Figure 3), the dominant species are the Eurasian 

(35.39%) and Pontic (26.40%) ones, followed at 

great distance by other categories of geoelements: 

European (8.98%), Central-European (6.17%), 

Mediterranean and sub-Mediterranean (6.17%), 

Balkan (5.61%), circumpolar (1.68%), Atlantic- 

Mediterranean, Taurean-Balkan, Carpatho-Balkan- 

Caucasian, and endemic (each with 0.56%). 

The large proportion of Pontic species reflects 

on the one side the dominance of the steppe 

meadows in the studied area, and on the other side, 

the nearness of the Razelm-Sinoe lagoon complex 

(located approx. 10 km east), which belongs to the 

Pontic biogeographic region. Among the categories

Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010) 

of Pontic elements (Fig. 4), the best represented in 

the studied area are: the Pontic-Mediterranean 

(55.31%), Pontic-Balkan (17.02%), Pontic proper 

(10.63%), Pontic-Pannonian-Balkan (8.51%), 

Pontic-Pannonian (4.25%), Pontic-Caucasian and 

Pontic-Central-European (2.12% each). The arid 

climate in the area of these hills is favorable for the 

large number of species of southern origin 

(Mediterranean, Sub-Mediterranean, Balkan), that 

make up a percentage of 11.78%. 

Table 2. The percentages of floristic elements 

in the studied area 

Floristic 

elements 

Sub- 

categories 

No. % 

Eua 25 

Eua Eua(Cont) 17 35,39 

Eua(Med) 21 

Eur 7 

Eur Eur(Cont) 3 8,98 

Eur(Med) 4 

SE Eur 2 

Euc 4 

Euc Euc -Med 5 6,17 

Euc- 1 

subMed 

Euc-Balc 1 

Pont 5 

Pont-Med 26 

Pont-Balc 8 

Pont Pont-Pan 2 26,40 

Pont-Pan- 4 

Balc 

Pont-Cauc 1 

Pont-Euc 1 

Med Med 10 6,17 

+ subMed subMed 1 

Balc 4 

Balc-Pan 2 

Balc Balc-Anat 1 

Balc-Cauc 2 5,61 

Balc-Pont- 1 

Anat 

47 

Taur- 

Balc 

- 1 0,56 

Carp- 

Balc- 

Cauc 

- 1 0,56 

Atl-Med - 1 0,56 

Circ - 3 1,68 

End - 1 0,56 

Balc 

5,61% 

Med 

6,17% 

Pont 

26,40% 

Cosm 

5,05% 

Adv 

2,24% 

Circ 

1,68% 

Others 

2,37% 

Euc 

6,17% 

Eur 

8,98% 

Eua 

35,39% 

Fig. 3. The spectrum of floristic elements 

Pont-Pan 

4,25% 

Pont-Pan-Balc 

8,51% 

Pont-Med 

55,31% 

Pont-Cauc 

2,12% 

Pont-Euc 

2,12% 

Pont 

10,63% 

Pont-Balc 

17,02% 

Fig. 4. The spectrum of the Pontic elements 

Among the ecological categories connected to 

soil humidity (Fig. 5), we can remark the 

considerable percentage of xero-mesophile (57.3%) 

and xerophile (24.71%) species, components of the 

steppe meadows and of rock formation vegetation. 

The mesophile species (14.6%) are present 

especially in the forested area of the hills. The 

eurythermal species have a smaller percentage 

(2.8%).


From the point of view of the preference for 

temperature (Fig. 5), the micro-mesothermal 

(46.62%) and moderately thermophile (37.64%) 

species have considerable percentages, as they are 

regular in the silvosteppe zone which is made up of 

steppe meadows and forests. The thermophile 

species (6.17%) are encountered either in the steppe 

meadows or in the rock formations. 

From the point of view of the preference for 

soil pH (Fig. 5), the higher percentages are held by 

poor acid-neutrophile (53.37%), acidic-neutrophile 

(17.97%) and euryionic species (20.22%). We must 

remark the high percentage of acidophile species 

(2.8%), grouped on the acidic volcanites that make 

up the rock formations in the plateau zone. 

70 

60 

50 

40 

% 

30 

20 

10 

0 

U% 

T% 

R% 

1-1,5 2-2,5 3-3,5 4-4,5 5-5,5 6 0 

ecological categories 

Fig. 5. The spectrum of ecological categories 

The 19 rare and endangered taxa (Table 3) 

represent 10.67% of the total species and 

subspecies identified in the Hills of Camena. A 

more important element is the presence of the 

endemic species Campanula romanica in the area, 

but also of other rare and very rare plants at 

national level, mentioned in the Red Book of 

vascular plants of Romania [11]: Dianthus 

nardiformis (Fig. 8), Silene compacta, Moehringia 

jankaea, Iris sintenisii, Salvia aethiopis, 

Sempervivum zeleborii, Galanthus plicatus, 

Nectaroscordium siculum subsp. bulgaricum, 

Achillea coarctata, Crocus reticulatus, etc. 

In terms of the main endangered categories 

(Fig. 6), 1 taxon (0.56%) is endangered, 4 taxa 

(2.24%) are vulnerable, while 14 other (7.86%) are 

rare, with small populations at national level. 

48 

Table 3. The rare and threatened taxa in the 

Camena Hills area 

No Name of the taxa Floris 

tic 

eleme 

nts 

1. Achillea coarctata Pont- 

Balc 

2. Allium flavum Taursubsp. 

tauricum Balc 

3. Campanula 

romanica 

4. Crocus reticulatus Pont- 

Med 

5. Dianthus 

nardiformis 

6. Echinops ritro 

subsp. ruthenicus 

IUCN 

categories 

[11] 

R - 

R - 

IUCN 

categories 

[10] 

End V/R EN 

V - 

Balc V/R VU 

Pont- 

Pan- 

Balc 

R - 

Balc R VU 

7. Galanthus 

plicatus 

8. Iris sintenisii Pont- 

Balc 

R LR 

9 Myrrhoides 

nodosa 

Med R - 

10 Moehringia Pont R VU 

. jankae 

11 Nectaroscordium Pont- R - 

siculum 

bulgaricum 

subsp. Balc 

12 Paeonia 

peregrina 

Balc V/R - 

13 Salvia aethiopis Pont- 

Med 

E/R - 

14 Sempervivum SE R - 

zeleborii 

Eur 

15 Seseli campestre Pont R - 

16 Silene compacta Pont- 

Med 

R EN 

17 Stipa ucrainica Pont- 

Cauc 

R VU 

18 Syrenia cana Pont R - 

19 Thymus zygioides Balc R -

NT 

89,33% 


E 

0,56% 

V 

2,24% 

R 

7,86% 

Fig. 6. The spectrum of sozological categories 


The research realized between 2008 and 2010 

led to the identification of 178 vascular taxa which, 

from the taxonomical point of view, belong to 46 

families and 38 orders. 

From the point of view of the biological forms, 

the dominant are the hemicryptophytes and 

terophytes, components of the steppe meadows in 

the area of Camenei Hills. The phanerophytes, 

camephytes and geophytes are present especially in 

the forested areas of these hills. Alongside the 

Eurasian species, well represented in the studied 

area are also the Pontic elements specific to west- 

Pontic steppes, but also those of southern origin 

(Mediterranean, sub-Mediterranean and Balkan), an 

expression of a climate with sub-Mediterranean 

nuances. 

Among the ecological categories of plants 

established according to their preference for 

substrate humidity, air temperature and soil pH, the 

predominant species are xero-mesophile, micromesothermal 

and moderately-thermophile ones, as 

well as the poorly acid-neutrophile ones. 

Of the total identified taxa, the rare and 

endangered species represent 10.67%. The 

important local populations of certain endemic and 

rare species at national level place the Camena Hills 

in the northern Dobrogea zones important from the 

conservation point of view. 

49 

5. References 

[1] PRODAN I., 1934-Conspectul florei Dobrogei 

I, Bul. Acad. de Înalte St. Agronomice, 

Tipogr. Naţională S.A., Cluj, 5, 1. 

[2] PRODAN I., 1935-1936 - Conspectul florei 

Dobrogei II, Bul. Acad. de Înalte St. 

Agronomice, Tipogr. Naţională S.A., Cluj, 6. 

[3] PRODAN I., 1938 - Conspectul florei Dobrogei 

III, Bul.Facult. de Agronomie, Cluj, Tipogr. 

Cartea Românească., 7. 

[4] SĂVULESCU T. (ed.), 1952-1976 - Flora 

României, vol. I-XIII, Edit.Academiei 

Române, Bucureşti. 

[5] DIHORU GH., DONIŢĂ N., 1970 - Flora şi 

vegetaţia podişului Babadag, Edit. Academiei 

R.S.R., Bucureşti. 

[6] CIOCÂRLAN V., 2000 - Flora ilustrată a 

României (Pteridophyta et Spermatophyta), 

Edit. Ceres, Bucureşti. 

[7] TUTIN T.G. HEYWOOD V.H., BURGES 

N.A., MOORE D.M., VALENTINE D.H., 

WALTERS S.M. & WEBB D.A. (eds), 1964- 

1980 - Flora Europaea, Vols. 1-5, Cambridge, 

Cambridge University Press. 

[8] TUTIN T.G. HEYWOOD V.H., BURGES 

N.A., MOORE D.M., VALENTINE D.H., 

WALTERS S.M. & WEBB D.A. (eds., assist. 

by AKEROYD J.R & NEWTON M.E.; 

appendices ed. by MILL R.R.), 1993 (reprinted 

1996) - Flora Europaea, 2 nd ed., Vol. 1, 

Cambridge, Cambridge University Press. 

[9] POPESCU A., SANDA V., 1998 - Conspectul 

florei cormofitelor spontane din România, Acta 

Botanica Horti Bucurestiensis, Edit. 

Universităţii din Bucureşti. 

[10] OLTEAN M., NEGREAN G., POPESCU A., 

ROMAN N., DIHORU GH., SANDA V., 

MIHĂILESCU S., 1994 - Lista roşie a 

plantelor superioare din România, Studii, 

Sinteze, Documente de Ecologie, Bucureşti, 

(1): 1-52. 

[11] DIHORU GH., NEGREAN G., 2009 - Cartea 

Roşie a plantelor vasculare din România, Edit. 

Academiei Române, Bucureşti.


Fig. 7. General aspect of volcanic hills of Camena 

Fig. 8. Dianthus nardiformis on the volcanic rocks 

of Camena 

50


Table 1. The list of the vascular plants of Camena Hills 

No. Taxa 

Family Life Floristic Ecological 

forms elements categories 

1. Achillea coarctata AST H Pont-Balc U1,5 T4,5 R4,5 

2. Achillea setacea AST H Eua(cont) U2 T3 R5 

3. Adonis flammaea RAN TH Pont-Med U2 T3,5 R3,5 

4. Agropyron cristatum subsp. POA H Pont-Euc U2 T4 R4,5 

pectinatum 

5. Agropyron ponticum POA H(G) Pont-Balc U1,5 T4,5 R4,5 

6. Ailanthus altissima SIM PH Adv U0 T0 R0 

7. Ajuga chamaepytis subsp. ciliata LAM TH Pont-Med U2,5 T4 R3 

8. Ajuga genevensis LAM H Eua(cont) U2 T3 R4 

9. Alliaria petiolata LIL TH-H Eua U3 T3 R4 

10. Allium flavum subsp. tauricum LIL G Taur-Balc U1,5 T4 R4 

11. Allium rotundum LIL G Euc(Med) U2 T4 R4 

12 Alyssum alyssoides BRAS TH Eua(Cont) U1 T3 R0 

13. Anagalis arvensis PRIM TH Cosm U3 T3,5 R0 

14. Androsace maxima PRIM TH Eua(Cont) U2 T4 R4 

15. Anemone sylvestris RAN H Eua(cont) U2 T3,5 R4 

16. Anthemis ruthenica AST TH Eur(Cont) U2 T4 R4 

17. Anthriscus cerefolium subsp. API TH Pont-Med U2,5 T4 R4 

trichosperma 

18. Artemisia absinthium AST H(CH) Eua U2 T3 R4 

19. Artemisia austriaca AST CH Eua(cont) U2 T4 R4,5 

20. Asparagus verticillatus LIL G Pont-Balc U1 T4,5 R4 

21. Asperula cynanchica RUB H Pont-Med U2 T3 R5 

22. Asperula tenella RUB H Pont-Balc U2 T4 R4 

23. Ballota nigra LAM H Euc U2 T3,5 R4 

24. Bassia prostrata CHEN CH Eua(cont) U1,5 T4 R4,5 

25. Berteroa incana BRAS TH Eua(cont) U2 T3 R4 

26. Brachypodium sylvaticum POA H Eua(Med) U3 T3 R4 

27. Bromus hordeaceus POA TH Eua U0 T3 R0 

28. Bromus sterilis POA TH Eua(Med) U2 T4 R4 

29. Bromus tectorum POA TH Eua(cont) U1,5 T3,5 R0 

30. Buglossoides arvensis BOR TH Eua U0 T0 R4 

31. Buglossoides purpurocaerulea BOR H-G Euc-subMed U2,5 T4 R4,5 

32. Calepina irregularis BRAS TH Pont-Med U2 T4 R3 

33. Camelina microcarpa BRAS TH Eua U3 T3 R0 

34. Campanula romanica CAMP H End U1,5 T4 R0 

35. Campanula sibirica CAMP H Eua(cont) U2,5 T4 R4 

36. Cardaria draba BRAS H Eua(Med) U2 T4 R4 

37. Carduus acanthoides AST TH Eur(Med) U2 T3 R0 

38. Carpinus orientalis CORY PH Balc-Cauc U3 T4 R4,5 

39. Carthamus lanatus AST TH Pont-Med U2,5 T4 R0 

40. Centaurea cyanus AST TH Med(Cosm) U3 T4 R0 

41. Centaurea diffusa AST TH Pont-Balc U2 T4 R0 

51


42. Cerastium brachypetalum CARY TH Med U3 T3 R0 

43. Chamomilla recutita AST TH Eua(Med) U2,5 T3,5 R5 

44. Chondrilla juncea AST H Eua U1,5 T3,5 R4 

45. Chrysopogon gryllus POA H Med U1,5 T4 R4 

46. Cichorium intybus AST H Eua U3 T0 R3 

47. Conium maculatum API TH-TH Eua U3 T3 R3 

48. Convolvulus arvensis CONV H(G) Cosm U2,5 T3,5 R3,5 

49. Convolvulus cantabricus CONV H Pont-Med U1,5 T3,5 R4 

50. Conyza canadensis AST TH Adv(Am.N) U2,5 T0 R0 

51. Cornus mas CORN PH Pont-Med U2 T3,5 R4 

52 Coronilla varia FAB H Eua(Med) U2 T3 R4 

53. Corydalis cava FUM G Euc U3 T3 R0 

54. Cotinus coggygria ANAC PH Pont-Med U2 T4,5 R4 

55. Crataegus monogyna ROS PH Eur U2,5 T3,5 R3 

56. Crepis sancta AST TH Pont-Balc U1,5 T4 R4 

57. Crocus reticulatus IRID G Pont-Med U2,5 T4 R3 

58. Crupina vulgaris AST TH Pont-Med U2 T3,5 R0 

59. Cynanchum acutum ASCL H Pont-Med U2,5 T4 R0 

60. Cynodon dactylon POA G(H) Cosm U2 T3,5 R0 

61. Daucus carota API TH Eua(Med) U2,5 T3 R0 

62. Dianthus nardiformis CARY CH Balc U1,5 T4,5 R4,5 

63. Dichanthium ischaemum POA H Eua(Med) U1,5 T5 R3 

64. Echinops ritro subsp. ruthenicus AST H Pont-Pan-Balc U1,5 T4 R4,5 

65. Elaeagnus angustifolia ELEG PH Adv 

66. Elymus repens POA G Circ U0 T0 R0 

67. Erodium cicutarium GER TH Cosm U2,5 T0 R0 

68. Eryngium campestre API H Pont-Med U1 T5 R4 

69. Erysimum diffusum BRAS H Eua(Cont) U1,5 T3 R4 

70. Euphorbia agraria EUPH H Pont-Med U2 T4 R0 

71. Euphorbia nicaeensis EUPH H Pont-Pan-Balc- 

Anat 

U1,5 T5 R5 

72. Festuca valesiaca POA H Eua(cont) U1 T5 R4 

73. Filipendula vulgaris ROS H Eua U2,5 T3 R4,5 

74. Fragaria viridis ROS H Eur(Cont) U2 T4 R3 

75. Fraxinus ornus OLE PH Med U1,5 T3,5 R5 

76. Fumaria rostellata FUM TH Euc-Balc U3 T0 R3,5 

77. Galanthus plicatus AMAR G Taur-Cauc U3 T4 R3 

78. Galium humifusum RUB H Pont-Balc U2 T4 R4,5 

79. Geranium divaricatum GER TH Eua(Med) U2,5 T3 R4 

80. Geranium pussilum GER TH Eur(Med) U2,5 T3 R0 

81. Geranium rotundifolium GER TH subMed U2 T3,5 R4 

82. Geum urbanum ROS H Eua(Med) U3 T3 R4 

83. Glechoma hirsuta LAM H(CH) Pont-Med-Euc U2,5 T3 R4 

84. Hieracium bauhinii AST H Euc U1,5 T3 R3,5 

85. Hieracium pilosella AST H Eua U2 T0 R2 

86. Holosteum umbellatum CARY TH Eua(Med) U2 T3,5 R0 

87. Hypericum perforatum HYP H Eua U3 T3 R0 

88. Iris sintenisii IRID G Pont-Balc U2 T4 R4 

52


89. Koeleria macrantha POA H Circ U2 T4 R5 

90. Lamium amplexicaule LAM TH Eua(Med) U2,5 T3,5 R0 

91. Lappula barbata BOR TH-TH Pont-Med U2 T3,5 R4 

92. Lathyrus tuberosus FAB H(G) Eua(Med) U2 T4 R4 

93. Ligustrum vulgare OLE PH Eua(Med) U2,5 T3 R3 

94. Linum austriacum LIN H Eua(cont) U1,5 T3,5 R4 

95. Malva sylvestris MALV TH(H) Eua(Cosm) U3 T3 R3 

96. Marrubium peregrinum LAM H Eua(Med) U2 T4 R0 

97. Marrubium vulgarae LAM H(CH) Eua(Med) U1 T4 R4 

98. Medicago lupulina FAB TH(H) Eua U2,5 T3 R4 

99. Medicago minima FAB TH Eua(Med) U1,5 T4 R4 

100. Melica ciliata POA H Eur(Med) U1,5 T4 R4 

101. Melilotus alba FAB TH Eua U2,5 T3 R0 

102. Minuartia setacea CARY CH Pont U1,5 T0 R4 

103. Moehringia jankae CARY H Pont U1 T4 R4,5 

104. Myosotis stricta BOR TH Eua(Med) U2 T0 R2,5 

105. Myrrhoides nodosa API TH Med U2,5 T4,5 R4,5 

106. Nectaroscordum siculum subsp. 

bulgaricum 

LIL G Pont-Balc U3,5 T3,5 R3,5 

107. Nigella arvensis RAN TH Pont-Med U2 T4 R4 

108. Nonea atra BOR TH Balc-Anat U2 T4 R3 

109. Onopordum acanthium AST TH Eua(Med) U2,5 T4 R4 

110. Onosma visianii BOR H Pont-Pan-Balc U1,5 T4,5 R4,5 

111. Origanum vulgare LAM H Med U2 T3 R3 

112. Orlaya grandiflora API TH Euc-Med U2 T3,5 R4 

113. Ornithogalum refractum LIL G Balc-Pan-Cauc U2 T3,5 R4 

114. Paeonia peregrina PAE H(G) Balc U2 T3,5 R5 

115. Papaver dubium PAP TH Eur U2 T3,5 R3 

116. Papaver rhoeas PAP TH Cosm U3 T3,5 R4 

117. Petrorhagia prolifera CARY TH Pont-Med U1,5 T4 R3 

118. Phleum phleoides POA H Eua(cont) U2 T3 R4 

119. Phlomis tuberosa LAM H Eua(Cont) U2,5 T3,5 R4 

120. Pinus nigra PIN PH Eua U0 T0 R0 

121. Plantago lanceolata PLAN H Eua U3 T0 R0 

122. Poa angustifolia POA H Eua U2 T3 R0 

123. Polycnemum majus CHEN TH Eua U1,5 T4,5 R4 

124. Polygonum aviculare POLG TH Cosm U2,5 T0 R3 

125. Polygonatum latifolium LIL G Pont-Pan-Balc U3 T3,5 R4 

126. Potentilla argentea ROS H Eua U2 T4 R2 

127. Potentilla recta s.l. ROS H Eur(Cont) U1,5 T3,5 R4 

128. Prunus mahaleb ROS PH Med U2 T3 R4,5 

129. Prunus spinosa ROS PH Eur(Med) U2 T3 R3 

130. Quercus petraea subsp. FAG PH E.Med.-Carp- U2,5 T2,5 R0 

dalechampii 

Balc 

131. Quercus pubescens FAG PH Med U1,5 T5 R5 

132. Ranunculus oxyspermus RAN H Balc-Cauc U2,5 T3 R3 

133. Reseda lutea RES TH(H) Eua(Med) U2 T3 R0 

134. Robinia pseudacacia FAB PH Adv(Am.N) U2,5 T4 R0 

53


135. Rosa canina ROS PH Eur U2 T3 R3 

136. Rumes acetosella subsp. POLG H SE Eur U2 T3 R2 

137. 

acetoselloides 

Salvia aethiopis LAM H Pont-Med U2 T5 R0 

138. Salvia nemorosa LAM H Pont-Med U2 T4 R4 

139. Salvia nutans LAM H Pont-Pan U1 T5 R5 

140. Sambucus nigra CAPR PH Eur U3 T3 R3 

141. Saxifraga tridactylites SAX TH Eua U2 T3,5 R4 

142. Scilla bifolia LIL G Euc U3,5 T3 R4 

143. Scleranthus annuus CARY TH-TH Eua U2 T3 R2 

144. Scleranthus perennis CARY H(CH) Eur U3 T0 R3 

145. Sedum maximum CRAS H Eur U2,5 T0 R4 

146. Sempervivum zeleborii CRAS CH SE Eur U1,5 T3,5 R4,5 

147. Senecio jacobaea AST H Eua U2,5 T3 R3 

148. Seseli campestre API H Pont U2,5 T4 R4 

149. Sideritis montana LAM TH Eua U2 T4 R4 

150. Silene compacta CARY TH Pont-Med U2 T4 R4 

151. Sisymbrium orientale BRAS TH Pont-Med U2,5 T4 R3 

152. Solidago virgaurea AST H Circ U2,5 T3 R3 

153. Sonchus oleraceus AST TH Cosm U3 T0 R0 

154. Stachys germanica LAM H Pont-Med U2 T4 R3 

155. Stachys recta LAM H Pont-Med-Euc U2 T5 R5 

156. Stipa capillata POA H Eua(Cont) U1 T5 R4 

157. Stipa ucrainica POA H Pont-Cauc U1 T4 R4 

158. Syrenia cana BRA TH Pont U1,5 T4 R4 

159. Teucrium chamaedrys LAM CH Euc(Med) U2 T4 R4 

160. Teucrium 

capitatum 

polium subsp. LAM CH Med U1,5 T4 R4,5 

161. Thalictrum minus RAN H Eua U2 T4 R4 

162. Thlaspi perfoliatum BRAS TH Eua U2,5 T3,5 R4,5 

163. Thymus pannonicus LAM CH Pont-Pan U1,5 T3,5 R4 

164. Thymus zygioides LAM CH Balc U1,5 T4 R4,5 

165. Tilia tomentosa TIL PH Balc-Pan U2,5 T3,5 R3 

166. Tragopogon dubius AST TH Euc(Med) U2,5 T3,5 R0 

167. Trifolium arvensae FAB TH Eua(Med) U1,5 T3 R4 

168. Trifolium campestre FAB TH Eur U3 T3 R0 

169. Trifolium echinatum FAB TH Med U1,5 T4,5 R4 

170. Urtica dioica URT H Cosm U3 T3 R4 

171. Valerianella lasiocarpa VAL TH Balc-Pont-Anat U1,5 T5 R4 

172. Verbascum phlomoides SCR TH Euc(Med) U2,5 T3,5 R4 

173. Veronica 

jacquinii 

austriaca subsp. SCR H Pont-Med-Euc U2 T4 R4 

174. Veronica teucrium SCR H Eua(Med) U1,5 T4 R4,5 

175. Vinca herbacea APOC H Pont U2 T5 R4 

176. Vincetoxicum hirundinaria ASCL H Eua(Cont) U2 T4 R4 

177. Viola kitaibeliana VIO TH Pont-Med U2 T4 R4,5 

178. Viola odorata VIO H Atl-Med U2,5 T3,5 R4 

54


IDENTIFICATION OF SOME ROSE GENITORS WITH RESISTANCE TO THE 

PATHOGENS AGENTS ATTACK 

*Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU and Elena DOROFTEI 

*Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology 

Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: mtrandafirescu@yahoo.com 

________________________________________________________________________________________ 

Abstract: In the South-Eastern Romania, as in all country the rose culture is higly praised for its ornamental 

value both in parks and domestic garden. In this zone of our country the rose culture is more important because 

the Black Seaside provide a better enviroment (82 km). Beside the growing of forign cultivars from Europe 

Companies, Romania has done a breeding work to develop autochtonous cultivars (Rusticana, Ambasador, 

Bordura de nea, Rosagold, Simina, etc.) better adapted to our local conditions. In rose breeding besides the 

ornamental value of these flowers (nice leaves, colours and shapes) the disease resistance has been taken into 

acount. Among the specific pathogens very harmful for the rose culture, one can mention: Sphaerotheca pannosa 

(Wallr) Lev var rosae Woron (powdery mildew), Diplocarpon rosae Wolf (black spott), Phragmidium 

mucronatum (Pers) Schlecht (rose rust) and Botrytis cinerea Pers (grey mold). One of the most effective methods 

to prevent these pathogens attack is breeding new cultivar and genically resistant genitors. These paper present 

the behaviour of 50 genotypes from rose collection of Fruit Growing Development of Fruit Tree Constanta and 

their response of such pathogens. The conditions of natural infections allowed grouping the biological material in 

4 classes of resistance. This clasifications was done acording to levels of frequency (F%) and intensity (I). The 

rose cultivars with genetic resistance to this pathogens are: Queen Elisabeth, Foc de tabara, Rubin, Parfum, 

Emerald d’or, Bel Ange, Apogee. 

Keywords: black spott, powdery mildew, rose rust, genitors, resistance 

__________________________________________________________________________________________ 


From the oldest times the rose was considered 

"Queen of The Flowers" due its beauty, perfume, 

richness in colors and multiple shapes of the grown 

cultivars. Therefore, the rose place is in the front of 

the ornamental species used for park and gardens 

decoration and for cut-flowers production. 

Unfortunately, the rose as many other cultivated 

plants can suffer due to the attack of some very 

damaging pathogens. Under favorable conditions, the 

pathogens can determine the partial or total 

defoliation of the plants, they become weak and the 

cut-flowers production can be diminished by quantity 

and quality as well. 

In order to prevent and control the pathogens 

specific to the roses, the studies carried out in 

Romania and in the World, were focused on 

identification of the species responsible for the 

diseases occurrence and knowledge of their biology 

(Bedian, 1980, Bernardis, 2004, Ostaciuc, 1982, 

Sandu 2004, Szekelly, 1981, Wagner, 2002), and on 

the other hand, was investigated the efficacy of some 

fungicides in order to control them(Bon and coll, 

1978, Morrison, 1978, Hagan and coll 1988, Losing, 

1988, Qvarnstrom, 1989, Raabe, 1989, Rolim and 

col, 1990, etc). 

The results obtained in the World (Saunders, 

1970; Klimenko, 1973; Simonyan, 1973; Semina, 

1980, 1984; Palmer, 1978; Costlediene, 1981) and in 

our country (Costache, 1993, Argatu, 1993, Sekely, 

1981, Wagner, 2002) clearly emphasized that the 

most efficient method to prevent the attack of the 

pathogens is the creation and extension in the culture 

of some roses cultivars genetically resistant to 

diseases. 

Therefore, the researches carried out during 

2008-2009 at Research Station for Fruit Growing 


Identification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) 

Constanta had as central objective the evaluation of 

some roses cultivars behavior to some key pathogens 

in order to identify some resistance donors genitors 

for further breeding works. 

In the reference area the pathogens of economic 

importance for the rose culture are Diplocarpon 

rosae Wolf, Sphaerotheca pannosa (Wallr)Lev var 

rosae Woron şi Phragmidium mucronatum (Pers) 

Schlecht. 


The biological material for investigations was 

represented by 50 roses cultivars preserved in the 

collection owned by Research Station for Fruit 

Growing Constanţa. Observation were made 

regarding the attack frequency (F%) and intensity (I 

notes) of the pathogens Diplocarpon rosa, 

Sphaerotheca pannosa var rosae and Phragmidium 

mucronatum, and finally the attack degree (AD) was 

calculated 

For disease intensity (I notes) the scale „0-6” 

was used. The observations were carried out in the 

period of attack maximum for each of three key 

pathogens studied. 

According the attack degree (A.D.) value, the 

cultivars were classified in five resistance classes as 

follow: 

- resistant (R) cu A.D.= 0-5% 

- slightly attacked (SA) with A.D. = 5.0-12.5% 

- medium resistant (MR) with A.D. = 12.5-22.5% 

- sensible (S) with A.D. = 22.5-37.6% 

- very sensible (VS) with A.D. = more than 37,6% 

To establish the D.L., in order to establish the 

five classes of cultivars the average value took into 

calculations ws 22.9%. 


In 2008, the attack of Diplocarpon rosae, fungi 

which determine the black spot disease was 

evidenced on rose cultivars in the third decade of 

May, being stimulated by the amount of precipitation 

at the vegetation start (114,9 mm) and by the high 

atmospheric relative humidity (over 75%). 

The disease symptoms occurrence on leaves 

consisted in some black spots, between 2-5 mm up to 

56 

10-18 mm in diameter, highly visible on the superior 

face of the leaves. (Fig.1). 

Fig. 1. Pătarea neagră a frunzelor de trandafir – 

Diplocarpon rosae 

Under such condition, presented in table 1, the 

(natural) genetic resistance to the pathogen attack 

manifested the cultivars: Emerande d’or, Grad 

Premiere, Traviata, Apogee, Luchian, Monica, Coup 

de Foudre and Tour Eiffel. 

The cultivars: Baccara, Pascali, Bel Ange, 

Creole, Dame de Coeur and Ingrid Bergman vere 

evidenced as slightly attacked (SA); and the cultivars: 

Flamenco, Karla, Rumba, Maria Callas, Grand Mogol 

and Montezuma were evidenced as medium resistant 

(MR). 

Very sensible (V.S) leaves black spot attack 

proved to be the cultivars Mainzer Fastnacht, Rose 

Gaujard, Kordes Perfecta, Detroit, Horido şi Konigin 

der Rosen, but they can be used as indicators for this 

disease. At this group of cultivars the plants were 

premature defoliated at the end of July. 

The climatic conditions from the Black Sea 

coast, characterized by strong wind, high temperature 

during the day (26-28 o C) and the presence of the 

water condense on the vegetative organs of the plants 

favorised, the occurrence of the powdery mildew 

attack produced by Sphaerotheca pannosa var rosae 

fungi starting with the last decade of May. 

The symptoms were noticed initially on the both 

sides of the leaves as irregular white dusty spots 

(Fig.2).

Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) 

Table 1. Behaviour of some cultivars roses to the 

attack of the main pathogens agents 

CULTIVARS Diplocarpon Sphaerotheca Phragmidium 

rosae pannosa var. rosae mucronatum 

G.A. Resistan G.A. Resistan G.A. Resistan 

(%) -ce class (%) -ce class (%) -ce class 

Brocade 7.2 S.A. 11.6 M.R. 12.3 M.R. 

Grand premiere 0 R 0 R 4.6 S.A. 

Creole 12.4 S.A. 6.5 S.A. 3.2 S.A. 

Traviata 0 R 1.2 R 14.6 M.R. 

Grand prix 32.4 S 0 R 0 R 

Horido 47.3 F.S. 1.0 R 3.6 R 

Apogee 0 R 0.8 R 0 R 

Luchian 0 R 4.2 S.A. 3.6 S.A. 

First love 1.2 R 17.6 M.R. 38.4 F.S. 

Concerto 3.7 R 9.7 S.A. 12.2 S.A. 

Konigin der 

Rosen 

63.0 F.S. 14.2 M.R. 0 R 

Foc de tabără 4.1 R 1.6 R 1 R 

Miss Univers 26.3 S 0 R 0 R 

Chicago Peace 32.0 S 37.6 F.S. 22.8 S 

Kronenburg 34.6 S 17.6 M.R. 0 R 

Bel Ange 4.3 S.A. 0 R 0 R 

Cocotte 0 R 0 R 18.3 M.R. 

Samurai 1.6 R 3.6 R 4.3 R 

Monica 0 R 0 R 0 R 

Montezuma 17.4 M.R. 9.6 S.A. 4.5 S.A. 

Don Juan 3.6 R 0 R 0 R 

Grand Mogol 15.0 M.R. 2.0 R 42.6 F.S. 

Sutter’s Gold 11.7 S 42.6 F.S. 53.2 F.S. 

Effel Tour 0 R 3.6 S.A. 1.2 R 

Detroit 37.9 F.S. 6.4 S.A. 0 R 

Maria Callas 15.6 M.R. 0 R 1.2 R 

Simfonia albă 24.0 S 0 R 1.2 R 

Mabella 57.0 F.S. 10.2 M.R. 13.7 M.R. 

Pascali 12.2 S.A. 0 R 0.9 R 

Rumba 19.3 M.R. 6.9 S.A. 22.0 M.R. 

King’s Ranson 0 R 1.2 R 54.6 F.S. 

Baccara 6.3 S.A. 22.0 S 7.2 S.A. 

Superstar 1.2 R 3.6 R 12.6 M.R. 

Kordes Perfecta 51.2 F.S. 17.9 M.R. 21.3 M.R. 

Madame 

Meilland 

0.7 R 7.3 S.A. 4.6 S.A. 

Coup Foudre 0 R 12.6 M.R. 17.2 M.R. 

Mr. Lincoln 2.6 R 0 R 0 R 

Dame de Coeur 7.2 S.A. 12.6 M.R. 42.1 F.S. 

Rose Gaujard 42.6 F.S. 51.8 F.S. 22.4 M.R. 

Carina 12.4 S.A. 6.3 S.A. 1.2 R 

Queen Elisabeth 3.2 R 1.2 R 0 R 

Eminance 1.2 R 2.6 R 0 R 

Mainzer 

Fastnacht 

38.6 F.S 7.9 S.A. 9.6 S.A. 

Karla 13.0 M.R. 1.6 R 2.0 R 

Flamenco 19.3 M.R. 3.6 R 12.3 S.A. 

Ingrid Bergman 7.2 S.A. 14.6 M.R. 1.2 R 

Ambassador 1.2 R 6.2 S.A. 1.6 R 

Emerande d’or 0.6 R 38.6 F.S. 0 R 

Parfum 0 R 1.8 R 2.6 R 

Rubin 0 R 0 R 0 R 

57 

Fig. 2. Făinarea trandafirului – Sphaerotheca 

pannosa var. rosae 

Afterwards, the attack progressed covering 

almost entirely the leaves, which turn in yellow, then 

dried and fallen down. In this case the attack 

progressed also on the young floral buds of the 

sensible cultivars, which were covered by the 

mycelium felt and they could not open. 

Assessment of the data presented in the same 

table revel that, a high (natural) resistance to this 

damaging pathogen stroke manifested the cultivars: 

Grand Prix, Miss Univers, Maria Callas, Pascali, 

Simfonia albă, their vegetative organs were entirely 

clear from Sphaerotheca pannosa var rosae. fungi 

symptoms. 

At the other pole were the cultivars: Emerande 

d’or, Chicago Peace, Sutter’s Gold and Rose Gaujard 

which were rated as very sensible (V.S.), but they can 

be used as sensibility indicators. 

The attack produced by Phragmidium 

mucronatum fungi, was noticed at the end of the first 

decade of May and progressed until the last decade of 

September. In this month this pathogen attack 

frequency (F%) and the intensity (I notes) registered 

the highest values. 

From the beginning the disease progressed on 

all plants organs: leaves, young branches, stalks and 

floral buds (Fig. 3). On these organs was noticed the 

presence of some orange pustules representing the 

fungus ecidia.

Identification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) 

Fig. 3. Rugina trandafirului – Phragmidium 

mucronatum 

In the last decade of May, on the inferior face of 

the plant leaves, small pale-yellow pustules occurs, 

representing the nest of uredospores which produce 

repeated secondary infections during the vegetation 

period. 

Starting with the second decade of June, on the 

inferior face of the plant leaves, was observed the 

presence of the black pustules representing the shelter 

of the teleutospores containing the resistance organs 

of the fungus. 

Among the cultivars that manifested a 

pronounced genetic resistance to the attack of this 

pathogen can be mentioned: Apogee, Bel Ange, 

Emerande d’or, Grand Prix, Kroenenburg, Kroningin 

der Roson, Miss Univers, Detroit, Rubin, Queen 

Elisabeth, Eminance. Their vegetative organs were 

entirely clear from pathogen signs all of the 

vegetation period. 

The vast majority of the other cultivar studied 

showed themselves as slightly attacked (SA) or 

medium resistant (MR). 

In the case of this pathogen, as was highlighted 

in table 1, very sensible cultivars (VS) manifested the 

cultivars Grand Mogol, Sutter’s Gold, King’s Ranson 

and First love. 


In the Romanian zone of Black Sea coast, the 

pathogens with economical importance for the roses 

grown in open fields are: Diplocarpon rosae Wolf, 

Sphaerotheca pannosa (Wallr) Lev var rosae Woron 

şi Phragmidium mucronatum (Pers) Schlecht. 

The rose cultivars Emerald d’or, Bel Ange, 

Apogee, Foc de tabără, Queen Elisabeth, Rubin, 

Parfum and Rubin, present genetic resistance for all 

three damaging agents and can be used as resistance 

genitors in the works carried out to bread new disease 

resistant rose cultivars. 

The fact that under the some climatic 

conditions, the rose cultivars manifest various attack 

degrees to the pathogens reveals that, the resistance is 

cultivars trait, which represent the key factor in 

prevention of the most damaging specific pathogens. 

58 

5. References 

[1] BEDIAN G., 1980. Rust (Phragmidium sp.) on 

roses, R.P.P., 59, 4, 1562. 

[2] BON Y., Bourdin J., Berthier G., 1978. Efficacité 

de quelques fongicides vis-á-vis de L’oidium du 

rosier (Sphaerotheca pannosa var. rosae), 

Phytiatrie – Phytopharmacie, 27 (3), 199-205. 

[3] CASTLENDINE P., Grout B.W.W., Roberts 

A.V., 1981. Cuticular resistance to Diplocarpon 

rosae, Transaction of the British Mycological 

Society, 47. 

[3] COSTACHE C., Costache M., Argatu Constanta, 

1993. Rezultate preliminare privind comportarea 

unor soiuri de trandafir la atacul principalilor 

agenţi patogeni. Analele I.C.L.F. vol. XII, 119- 

129. 

[4] HAGAN A. K., Gillian C. H., Fare D. C.,1987. 

Evaluation of new fungicides for control of rose 

black spot, Journal of Environmental Horticulture 

6 (2), 67-69. 

[5] LÖSING H., 1988. Bekämpfung von Rosenrost, 

Deutsche Baumschule 40 (11), 518-519. 

[6] MORRISON L. S., 1978. Preliminary results on 

the evaluation of fungicides for the control of 

black spot of rose. Nursery Research Field Day P 

– 777, 59-60. 

[7] QVARNSTRÖM K., 1989. Control of black spot 

(Marssonina rosae) on roses, Växtskyddsnotiser 

53 (3), 58-63. 

[8] PALMER L. T., Salac S. S., 1978. Reaction of 

several types of roses to black spot fungus, 

Diplocarpon rosae, Indian Phytopathology 30 

(3), 366-368. 

[9] ROLIM P. R. R., Toledo A. C. D., Cardoso R. 

M. G., Brignani Neto F., Oliveira D. A., 1990. 

Comparison of fungicides for control of rose 

black spot (Diplocarpon rosae) and powdery 

mildew (Sphaerotheca pannosa var. rosae), 

Summa Phytopathologicals 16 (3-4), 269-274. 

[10] SAUNDERS P. J. W., 1970. The resistance of 

some cultivars and species of Rosa to 

Diplocarpon rosae Wolf causing black spot 

disease, Natn. Rose, A., 118-128. 

[11] SEMINA S. N., Klimenco Z. K., 1976. 

Evaluation of garden rose gene pool for

Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010) 

resistance to powdery mildew. Byull Gosudar, 

Nikit. Bot. Sada, 2 (30), 48-54. 

[12] SIMONYAN S. A., 1973. Powdery mildew of 

rose in the Erevan Botanical Grden. Biol. J. 

Armenii, 26 (7), 62-73. 

[13] SZEKELY I., Wagner Şt., Drăgan Maria, 1981. 

Rezistenţa diferitelor soiuri de trandafir faţă de 

atacul de făinare (Sphaerotheca pannosa var 

rosae) în funcţie de unele caracteristici anatomomorfologice, 

Simpoz. CAER Cluj, ASAS, ICPP. 

[14] WAGNER Şt., Râureanu V., 1996. Principalele 

boli şi dăunători ai trandafirilor şi combaterea 

lor. Rosarium, nr. 1. 

59


PRELIMINARY DATA ON MELEDIC – MANZALESTI NATURAL RESERVE (BUZAU 

COUNTY, ROMANIA) 

Daciana SAVA *, Mariana ARCUŞ**, Elena DOROFTEI * 

*Ovidius University, Natural Sciences Faculty, 

Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania, e-mail: daciana.sava@gmail.com 

** Ovidius University, Faculty of Pharmacy, Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania 

__________________________________________________________________________________________ 

Abstract: the Meledic –Manzalesti Reserve is situated in the central-eastern part of Romania, in Buzau County, 

60km north form town of Buzau. The Reserve (136 hectares) is delimitated by four rivers and it is situated at a 

medium altitude of 530 m. Because of the remarkable forms of relief, appeared as a result of dissolution of salt, the 

presence of a salt cave unique in Europe and of a number of lakes with fresh water, this area was declared in 1986 

Geological and Speological Reserve. Later (in 2000) due to the presence of an interesting flora and fauna it was 

established the value of its natural heritage, and was declared as „Protected Natural Area” with geological, 

speological, floral and faunistic importance. In 2007 it was declared „Site of Community Importance” and will 

become area of special conservation after the validation of the European Commission. The present study took place 

over a period of two years, with field trips in various periods of the year. As for the flora, taxons belonging to over 

100 genera were identified, Most genera belong to Fabaceae, Asteraceae, Labiatae, Rosaceae and Umbelliferae 

families. The statistical analysis showed as biological forms, the predominance of hemicrytophytes. As floristic 

elements, the Euro-Asiatic and Central European elements predominated. As regarding the ecological preferences 

(humidity, temperature and soil reaction) it has been observed the domination of xeromesophytic, mezothermal and 

euriionical species. 

Keywords: Natural Reserve, Manzalesti –Meledic Natural Reserve, Romania 

__________________________________________________________________________________________ 


In 1978, a group of Romanian spelologists, part of 

“Emil Racoviţã” Speologists Club from Bucharest, 

discover in the sub-Carpathians Mountains at 

Mânzãleşti, the longest (300 m), the deepest (44 m) 

and the most ramificated cavity in salt in the country, 

second in the world as oscillation of level, third as 

length, wich they named “The cave with three 

entrances“ from Sãreni. In 1980 the “6s“ Cave from 

Mânzãleşti is discovered, the longest cave in salt 

world-wide at that moment (1257 m) and the second as 

oscillation of level (-32 m), with numerous 

ramifications. Later on, other galleries, were 

discovered of a total of 4257 m length, 32 caves 

digged in salt, taking the Mânzãleşti cave to the 

second place in the world for caves digged in carst of 

salt. 

Later on, in 1986, the salt carst from 

Mânzãleşti becomes a reserve of The Romanian 

Academy from the geological and speleological point 

of view. 

According to the Habitat Directive 92/43/CEE 

concerning the conservation of natural habitats, wild 

flora and fauna, the protected areas network Natura 

2000 appears in România, which includes also in this 

network the Meledic Plateau of Mãnzãleşti commune, 

Buzãu County, according to Law nr 5/5 March 2000 

[1]. 

The site has the ROSCI 0199 code and is 

classified under category IV (according to UICN) as 

Special Conservation Area. The reserve is part of the 

Continental Biogeographical Region; its existance is 

trying to protect the “Ponto-sarmatic deciduous 

thickets” habitats. 

Characterization of the “Meledic Plateau” 

Reserve 


Preliminary data on Meledic-Manzalesti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) 

„Meledic Plateau” Reserve is situated in the sub- 

Carpathians Mountains, in the Lopãtari Dingle and in the 

Slãnic river superior basin (tributary streamof Buzãu 

river) (latitude N 45 ο 29’ 49” and longitude of E 26 ο 37’ 

16”). The reserve has a length of 1.7 km, on the North- 

South axis and 1.2 km on the West-East direction, 

holding a total surface of 157 hectares, the plateau being 

situated at an altitude between 400m and 600m. 

In the Eastern side, the delimitation is determined by 

the Jgheab river (a tributary stream of the Slãnic river), 

North of the Meledic stream and West of the Sãrat 

stream, the latter tributary stream of the Jgheab Valley, 

which emptyes into the Sãrat stream in turn. Out of these 

rivers and streams only the Meledic stream has fresh 

water, the other streams being also alimented with salty 

springs, their water having a brackish taste. 

Relief 

The Meledic Plateau Slopes are very abrupt, 

allowing sometimes to see the structure of the plateau 

represented by a layer of clay and shale on the upper 

side, with a thickness of 10 to 30 meters, under which 

there is the block of salt, tall up to a few hundred meters. 

The Meledic Reserve represents one of the most 

unprecedented places, the relief is expanding on the salt 

located on the surface or shallow depth, resulting one of 

the most interesting regions in our country. A very 

diversified terrain in shape and size develops because of 

the dissolution of salt on slopes (Fig.1, Fig.2). 

Fig.1. Aspect of the abrupt slopes in Plateau 

Meledic Reserve (south view) 

62 

Fig.2. Aspect of the abrupt slopes in Plateau 

Meledic Reserve (vest view) 

On the western side we can notice blocks of salt 

integrated in clay and salty shale, on which gaps and 

limestones have developed, in comparison with the 

southern side where vein of salt can be noticed even on 

the surface. Where the salty water rivers come out on 

the surface arises a rapid vaporisation of the water 

resulting in especially beautiful salt cristals. 

The plateau is located on the upper side of the 

reserve and is crossed by sinkholes, closed dingles, 

oval or round, with a diameter that can reach 

sometimes 40 m and a depth of 25 m, wider dingles 

results by their blending. 

On the bottom of such sinkholes, where the salt 

was covered with a denser layer of clay, freshwater 

lakes were formed, receiving water only from rain or 

snow meltdown. These lakes have karstic origins, all 

the underground springs are salt watered, the 

connection with these ending long time ago. The 

presence of freshwater lakes on a salt massif is 

considered a unique phenomenon. 

Soils 

In the Meledic reserve we encounter a large 

variety of soils. 

On the steep slopes, where the salt layers are very 

close to the surface or even on the surface, we find the 

white alkali. On the slopes where the water carries 

small amounts of silt the vertisols are formed, on 

heavy clay rocks (with high clay content). 

On the plateau we meet halomorphous soils, 

which have a high content of soluble salt, that occur on

Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010) 

partially covered with sediment surfaces. A great 

diversity of halophile species develop here. 

In sinkholes where the sedimented clay have 

allowed the formation of lakes, the soils are 

hydromorphic, formed due to an excess of moisture, 

which can be permanent or temporary. In areas where 

the water has a permanent stagnation, the pseudogleic 

soil is formed. The plant species developing here are 

thermophilic. 

In forested areas we can find reddish brown 

forest soils, especially clay soils. In these areas we 

especially find Pontic or Mediterranean species. 

Climate 

The sub-Carpathians Mountains have a 

temperate-continental climate, with regional 

differences imposed by the shape of relief, but also by 

the position at the intersection of climate influences 

northwest, eastern and southern. 

Being located in a lowland area of the sub- 

Carpathians, the Meledic Plateau has a low hill 

climate with a tendency of aridity in summer. The 

average annual temperatures fit between 6 ο C and 8 ο 

C. The average annual temperature of the coldest 

month, January, is of 3 ο C, and of the hottest month, 

July, is of 18 ο C. 

The average annual rainfall is around 700-800 

mm. The largest amount of rainfall is in May and 

June, and the driest months are September and 

October. 


Field trips have been organized for the flora 

studies in the Meledic Reserve from Mânzãleşti: two 

trips in the months of May and June (months that 

have a rapid vegetation development), and in the 

months of April, July, August, September only one 

trip, through the years 2007-2008 to catch the 

different stages of vegetation (vernal and estival). 

After rating, we made up a floristic list, in which 

the plants have been placed in the right systematic 

units [1, 2, 3]. Based on this list, we made out: the 

systematic analysis of the vegetation, the bioform 

spectrum, the geoelements spectrum, the spectrum for 

ecological preferences: humidity, temperature and 

soil reaction [4, 5]. 

63 


Due to the field trip a total of 133 taxa were registered. 

The following taxa were identified in the study area: Acer 

campestre L. Ph (MM); Eur.; U2,5 T3 R3, Achillea 

millefolium L. H; Euras.; U4 T3 R0 , Adonis aestivalis L. 

Th; Euras; U3T4 R3, Agrimonia eupatoria L. H; Euras.; U2,5 

T3 R4, Ajuga genevensis L. H; Euras.; U2,5 T3 R4 , Alisma 

plantago-aquatica L. HH; Cosm.; U6 T0 R0, Alnus incana 

(L) Moench Ph (MM); Eur.;U4 T2 R4, Alnus viridis 

(Chaix.) DC Ph (MM);Alp.-eur;U3,5 T2,5R3, Anchusa 

officinalis L. limba boului); TH; Eur.; U2 T3,5 R0, Anemone 

nemorosa L. G; Eur; U3,5 T4 R0, Anemone ranunculoides 

L. G; Eur; U3,5 T3 R4, Artemisia vulgaris L. H; Circ.;U3 T3 

R4, Astragalus onobrychis L. H; Euras.; U1,5 T3,5R4,5, 

Ballota nigra L. Th; Centr. Eur.); U3 T3,5 R0, Betonica 

officinalis L .(Stachys officinbalis L.) H; Euras.; U3 T3 R3, 

Brassica rapa L.Th; Med; U3 T3 R4, Campanula 

rapunculoides L. H; Euras.; U3 T2 R0, Capsela bursapastoris 

Medicus Th; Cosm; U3 T0 R0, Carex digitata L. 

H; Euras.; U3 T3R3, Carum carvi L. TH; Euras.; U3,5 T3 R3, 

Centaurea spinulosa Roch. H; Centr. Eur.; U2,5 T0 R3, 

Centaurea nervosa Willd. H; Alp.-eur.;U3 T0 R3; 

Centaurium umbellatum Gilib. Th; Centr.eur.;U3 T3 R2, 

Chaerophyllum bulbosum L. TH; Centr. Eur; U4 T3,5 R4,5, 

Chrysanthemum leucanthemum L. H; Euras.; U 3T3,5 R3, 

Chrysanthemum corymbosum L. H; Euras.; U3 T3 R3, 

Clematis vitalba L. Ph ; Centr. Eur.; U3 T3 R3, Colchicum 

autumnale L. G; Eur; U3,5 T3 R4, Coronilla varia L. H; 

Centr. Eur.; U2 T3 R4, Cornus mas L. Ph (M); Pont. medit.; 

U2 T3,5 R4, Cornus sanguinea L. Ph (M);Centr. Eur); U3 T3 

R4, Corylus avellana L. Ph (M); Eur.; U3 T3 R3, Crataegus 

monogyna Jacq. Ph (M); Euras.;U2,5 T3 R3, Cytisus 

hirsutus L. Ph (N); Centr. Eur.; U2,5 T3 R2, Daucus carota 

L. TH; Euras.; U2,5 T3 R0, Delphinium consolida S.F.Gray 

(Consolida regalis) Th; Euras; U3 T4 R4, Diplotaxis muralis 

L. Th; Centr. Eur; U2,5 T3,5 R4, Draba verna Chevall Th; 

Euras; U2,5 T3,5 R0, Echium vulgare L. TH; Euras.; U2 T3 

R4, Elaeagnus angustifolia L. Ph (M); Euras; U0 T3 R4,5, 

Epipactis atropurpurea Raf G; Euras.; U2 T0 R4,5, 

Equisetum arvense L. G.; Cosm.; U3 T3 R0, Erigeron 

canadensis L. Th; Adv.; U2,5 T0 R0, Eryngium campestre 

L. H; Pont. medit.; U1 T5 R4 , Euphorbia cyparissias L. H; 

Eur.; U2 T3 R4, Euphrasia rostkoviana Hayne. Th; Centr. 

Eur.; U3 T3 R3, Fagus sylvatica L. Ph (MM); Centr. Eur.; 

U3 T3 R0, Festuca pratensis Hudson H; Euras; U3,5 T0 R0, 

Ficaria verna L. (Ranunculus ficaria Huds.) H;Euras: U3,5


T3 R3, Filipendula ulmaria Maxim. H; Euras: U4,5 T2 

R0, Fragaria viridis Duch. H; Euras.; U2 T4 R3, 

Fraxinus ornus L. Ph (M); Medit.; U1,5 T3,5 R5, Gagea 

pratensis Dumort. G; Eur; U2 T3 R3, Galanthus nivalis 

L. G; Centr. Eur.; U3,5 T3 R4, Galium verum L. H; 

Euras.; U2,5 T2,5 R0, Galium vernum Scop. H; Euras; U3 

T2 R2, Hippophae rhamnoides L. Ph (M); Euras.; U0 T3 

R4,5, Hypericum perforatum L. H; Euras.; U3 T3 R0, 

Juniperus communis L. Ph (M); Circ.; U2 T0 R0, 

Knautia arvensis (L.) Coult. H; Eur.; U2,5 T3 R0, 

Knautia silvatica Duby. H; Centr. Eur; U2 T3 R0, Larix 

decidua Miller Ph (MM); Carp; U2,5T0 R0, Lathyrus 

tuberosus L. H; Euras.; U2 T4 R4, Lathyrus pratensis L. 

H; Euras; U3,5 T3 R4, Leontodon hispidus L. H; 

Euras.;U2,5 T0 R0, Lepidium draba Desv. H; Euras: U2 

T4 R4, Linum austriacum L. H; Euras.; U1,5 T3,5 R4, 

Lithospermum purpureo-caeruleum L. H; CentrEur;U2 

T3,5 R4, Lythrum salicaria L. H; Circ.; U4 T3 R0, 

Medicago lupulina L. Th ; Euras.; U2,5 T3 R4, Medicago 

falcata L.Th; Euras.; U2 T3 R4, Melampyrum arvense L. 

Th; Eur.; U2 T3,5 R4,5 , Melilotus officinalis (L.) Pallas 

Th; Euras.; U2,5 T3,5 R0, Morus nigra L. Ph (MM); Adv; 

U2 T3,5 R4, Muscari comosum (L. ) Miller G; Eur.; U1,5 

T3,5 R0, Myosotis sylvatica Hoffm. H; Euras: U3,5 T3 R3, 

Onobrychis viciifolia Scop.H; Euras.; U2 T3 R0 , Orchis 

purpurea Huds. G; Centr. Eur.;U 2,5 T4 R4,5, Origanum 

vulgare L. H; Euras.;U2,5 T3 R3, Orlaya grandiflora L. 

Th; Med; U2 T3,5 R4, Ornithogalum refractum Kit. G; 

Balc-Pan- Cauc; U2 T3,5 R4, Phragmites australis 

Steudel HH; Cosm.; U5 T0 R4, Picea excelsa Link Ph. 

(MM); Centr. Eur.; U0 T0 R0, Pinus sylvestris L. 

Ph.(MM); Euras.; U0 T0 R0, Plantago media L. H; 

Euras; U2,5 T0 R4,5, Poa pratensis L. H; Circ: U3 T0 R0, 

Polygala amara L. H; Eur.; U0 T2 R4,5, Polygala major 

Jacq. H; Pont. –medit.; U2 T3 R4,5, Potamogeton natans 

L. HH; Cosm.; U6 T 2,5 R4, Potentilla reptans L. H; 

Cosm; U3,5 T0 R4, Potentilla argentea L. H; Euras; U2T4 

R2, Primulla officinalis Hill. H; Euras.;U3 T2 R5, 

Prunella vulgaris L. H ; Circ. U3 T3 R0, Prunella 

grandiflora (L.) Scholler H; Eur.; U3 T3 R4,5, Prunus 

cerasifera Ehrh. Ph (M); Euras: U2 T4 R0, Pyrus 

piraster Burgsd. Ph (M); Eur; U2 T3 R4, Quercus 

dalechampii Ten. Ph (MM); Medit; U2,5 T3 R0, 

Ranunculus arvensis L. Th; Euras.; U3 T3 R0, 

Rhinanthus minor L. Th; Eur.; U3 T0 Ro, Rosa canina 

L. Ph (N); Eur.; U2 T3 R3, Rubus caesius L. Ph (N); 

Eur.; U2 T3 R4, Salix alba L. Ph (MM); Euras; U5 T3 R4, 

Salix caprea L. Ph (M); Euras; U3 T3 R3, Salix 

64 

pentandra L. Ph (MM); Euras.;U4,5 T0 R3,5, Salvia 

verticillata L. H; Medit.;U2 T4,5 R4, Salvia nemorosa L. H; 

Centr. Eur.; U2,5 T4 R3, Scabiosa ochroleuca L. H; Euras.; 

U2 T4 R4, Schoenoplectus tabernaemontani (Gmelin) 

Palla HH;Euras.; U5,5 T4 R5, Senecio vernalis Waldst et. 

Kit. 

Th; Euras.;U2,5 T4 R0, Silene vulgaris Garke H; Euras: U3 

T3 R4, Sinapis arvensis L. Th; Euras.; U3 T4 R4, 

Sisymbrium sophia Webb. Th; Euras; U2,5 T4 R4, Stachys 

lanata Jacq. H; Medit.; U2 T0 R0, Thlaspi perfoliatum L. 

Th; Euras; U2,5 T3,5 R4,5, Thymus glabrescens Willd. Ch; 

Pont.-pan.; U2 T4 R0, Tilia cordata Miller Ph (MM); Eur.; 

U3 T3 R3, Tragopogon pratensis L. H; Euras.; U3 T2 R3, 

Trifolium campestre Schreb. Th; Eur; U3 T3 R0, Trifolium 

medium L. H; Euras.; U3 T3 R0, Typha angustifolia L. 

HH; Circ.; U6 T4 R0, Ulmus laevis Pall. (velniş); Ph (MM); 

Eur.; U4 T3 R3, Veronica chamaedrys L. Ch; Euras.; U3 T0 

R0, Veronica arvensis L. Th; Eur; U2,5 T3 R,Veronica 

teucrium L Ch; Euras.; U1,5 T4 R4,5, Vicia angustifolia L. 

Th; Euras.; U2 T3 R 0, Vicia sepium L. H; Euras.; U3 T3 R3, 

Vicia cracca L. H; Euras; U3 T0 R3, Vicia hirsuta S.F. 

Gray. Th; Euras; U2.5 T3,5 R4, Viola arvensis Murr. Th; 

Cosm.; U3 T3 R0, Viola hirta L. H; Euras.; U2 T3 R4, Viola 

tricolor L. Th; Euras.; U2,5 T3 R0. 

Statistic flora analysis 

Taxa found in the reserve belong to 4 classes, 43 

families and 133 species. The largest number of species has 

the following families: Fabaceae (16 species), Labiatae, 

Rosaceae and Compositae (10 species each), 

Scrophulariaceae, Umbelliferae (5 species each), the other 

families were represented by only 1, 2, or 3 species each. 

Analysis of the biological forms 

Analyzing the spectrum of the biological forms we 

discover that in the reserve hemicryptophyte dominate 

(41%) from the species identified. These are followed 

(21%) by the phanerophytes which together with therophites 

(24%) form another 43% from the biological forms, the rest 

being represented by the geophytes and the helohydrophytes 

(Fig.3).

4% 

8% 

20% 

2% 


4% 

21% 

41% 

H Ph Ch G HH Th TH 

Fig.3. Analysis of the biological forms 

Analysis of the floristic elements 

The analysis of the floristic elements mark out the 

dominant euro Asiatic elements, which among those 

central European sum up approximately 82 species 

(70%) from the reserve flora, forming more than half the 

floral elements which means that they constitute the 

floristic background of this reserve. 

Mediterranean and Ponto Mediterranean floristic 

elements, which are thermophile species found especially 

on the sunny slopes, form together 8%. However, the 

cosmopolitic species are also remarkable, representing 

5% from the total species found in the reserve. 

The fact that the reserve in situated in sloppy area is 

confirmed by the presence of the circumpolar and even 

alpine European at the reserve level, together 

representing 7% of the total species (Fig.4). 

46% 

1%1%1% 5% 

5% 

2% 

1% 

5% 

2% 

14% 

14% 

Daco.Balc Carp. Balc.Pan. Cauc Circ 

Cosm Adv Eur Centr.Eur 

Alp Pont Pan Medit Euras 

Fig.4. Analysis of the floristic elements 

Ecologic study of the cormophytes 

Humidity 

If we group the plants by their humidity regimen in 

which they are adjust to live here, we will discover that 

the most dominant are xeromesophilic (U2-U2,5) which 

65 

are 42% from the total of the identified species in the 

reserve, these being found in the droughtiest places, 

specially on the meadows. 

Notable are also the mesophilic (U3-U3,5) which have 

a percentage of 37%, and can be found in areas where the 

light is scarce or there is an excess in humidity, where the 

swamps dry up during the summer but nevertheless have an 

excess in moisture. 

The xerophilic (U1-U1,5) can be found in 5% and this 

shows the hot and arid summer climate, being especially 

noticed on the slopes that have a south exposure, covered 

with a small seam of clay soil. 

In a percentage of 6%, the mesophilical (U4-U4,5) 

species that prefer soils from humid to moist-wet, are found 

near lakes or where there is an excess in moisture all year 

long. 

Remarkable is the presence of the hydrophilic (U5- 

U5,5) and ultrahydrophilic (U6) species which together 

form 5% from the total of species, and can be found in the 

ponds or on their border where water is present all year 

round. 

The amphytoletant (U0) species can also be found in 

the reserve in a percentage of 6%, being the most adaptable 

for these special conditions (Fig.5). 

22% 

20% 

4% 

1% 

5% 

2% 1% 2% 2% 

4% 

28% 

U3 U3,5 U4 U2,5 U5 U5,5 U6 U0 U1 U1,5 U2,5 U2.5 

Fig. 5. Ecological spectrum of humidity 

Temperature 

Mesothermal (T3-T3,5) appear in a percentage of 

63%. Mild thermophilic (T4-T4,5) species appear in a 

percentage of 12%, which suggests that the climate in the 

reserve in a temperate-continental one.The 

amphylotolerant (T0) appear in 16% of the total. 

The cryophilic (T1) species are missing, and the 

microthermal (T5) species appear in a small percentage, 

only 1% (Fig.6). 

9%


45% 

2% 

6% 

17% 

1% 1% 

14% 

45% 

14% 

T3 T3,5 T4 T4,5 T5 T0 T2 T2,5 T3 

Fig.6. Ecological spectrum of temperature 

Soil reaction 

37% of the identified species are euroionic (R0); 

poor acid-neutrophilic species (R4-R4,5) are 

presented in the same proportion; acid-neutriphilic 

species (R3-R3,5) can be found in a percentage of 21%, 

and the neutrophilic-basophilic (R5) are found only in a 

percentage of 3%. The balance of the acidophilic plants 

(R2) is just 2%, and those highly acidophilic (R1) are 

missing (Fig.7). 

34% 

2% 

10% 

3% 

20% 

R3 R3,5 R4 R4,5 R5 R0 R2 

30% 

Fig. 7. Ecological spectrum for soil reaction 


The flora in the reserve is highly diversified, being 

represented by the distribution of the species in 43 

families, predominant being the families Fabaceae, 

Labiatae, Compositae. 

Hemicryptophyte (41%) appear in the highest 

percentage indicating the presence of the herbal 

evergreen species, adaptable to the edapho-climatic 

conditions in the areas. Therophites (20% + 4%) are 

1% 

66 

plants mostly found in the northern regions, with an arid 

climate, and are presented in the reserve through the 

annual or biannual species. Phanerophytes (21%) 

presented by trees and scrubs, indicate the presence of 

forests in the reserve, as well as the cover of the slopes 

with scrubs which assure its stabilization. 

The analysis of the floristic elements reveals the 

predominant Euro-Asian elements, which along the central 

European totalize approximately 82 species (70%) from 

the reserve flora, representing the floristic background of 

the reserve. Floristic Mediterranean and Ponto 

Mediterranean elements, which are theomophilic species, 

can be found on 

the sunny slopes. The fact that the reserve in situated in a 

hill area can also be acknowledged because of the 

circumpolar and even alpine European species found here. 

The presence of xeromesophitic species (U2-U2,5) in a 

percentage that represents almost half of the total of the 

identified species in the reserve (42%), indicate a arid 

climate which is specially caracteristical for the medows. 

The mesophilic species (U3-U3,5 ), which have a pretty 

high percentage (37%), can be found in the areas where the 

light is scarce or it is excessively moisturized or in the 

areas where the swamps completely dry out during 

summer, but remain excessively moisturized. The 

mesothermal (T3-T3,5) along the mild thermophilic (T4- 

T4,5) are presented in a higher percentage, which mean that 

the climate in the reserve is a temperate continental one. 

Regarding the distribution according to the reaction of the 

soil, the euroionic species (R0) can be found in a pretty 

high percentage (34%), almost equal to those of the 

species and the poor acid-neutrophilic (R4- R4,5) (40%). 

Remarkable is also the presence of the acid-neutrophilic 

species (R3- R3,5) (21%) which have a percentage that is 

worth taking into account; the presence in the reserve of 

different types of habitats: rivers (with fresh and salt 

water), lakes, meadows, forests. 

Studies are to be done in the future to analyze the 

interesting and diverse flora of the region. 

5. References 

[1] MOHAN GHE, ARDELEAN A., GEORGESCU M., 

1993 - Rezervaţii şi monumente ale naturii din 

România, Casa de Editurã şi Comerţ, București, 201 

pp.


[2] BELDIE AL., 1977- Flora României - 

Determinator ilustrat al plantelor vasculare, vol. I- 

II, Editura Academiei R.S.R, 406 pp. 

[3] CIOCÂRLAN V., 2000 - Flora ilustratã a 

României, Editura Ceres, Bucureşti, 1138 pp. 

[4] DONIŢÃ N., IVAN D., 1975 - Metode practice 

pentru studiul ecologic şi geografic al vegetaţiei: 

112-331, Editura Didacticã şi Pedagogicã, 

Bucureşti. 

[5] SANDA V., POPESCU A., DOLTU I., DONIŢÃ 

N., 1983 - Caracterizarea ecologicã şi 

fitocenologicã a speciilor spontane din flora 

României, “Ecological and phytocoenologyical 

characterisation of the spontaneous species in 

Romanian flora” in: Nat.Scienc.Suppl. 25, 

Stud.Communic. Muz. Brukental, Sibiu, 126 pp. 

67


CONTRIBUTIONS TO THE BIOMETRICAL AND PHYTOBIOLOGICAL STUDY 

ON WILD GARLIC 

Mariana LUPOAE*, Dragomir COPREAN*, Rodica DINICĂ**,Paul LUPOAE*** 

* Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences 

Street Mamaia, nr. 124, Constanţa, 900527, România, mariana_lupoaie@yahoo.com 

** Dunărea de Jos University Galaţi, Faculty of Science, Street Domnească no. 47, 800008, Galaţi, 

rodinica@ugal.ro 

*** Natural Sciences Museum Complex Galaţi-Botanical Garden, Street Regiment 11 Siret no. 6A, 800340 

Galaţi, paul_lupoae@yahoo.com 

_____________________________________________________________________________________ 

Abstract: The purpose of our study was the biometrical and phytobiological analysis of leafs and bulbs on wild 

garlic. This species grows spontaneously in the Romanian flora and was harvested for obtaining the drugs on 

Măcin Mountains (Luncavita Forest), at altitudes of 150÷200m. By macroscopic examinations in different 

phenophasis established in the area study extended population with Allium ursinum L. ssp. ucrainicum Kleopow 

et Oxner (Fam. Alliaceae). The biometrical calculation have been performed according to the literature, early 

spring, in months february, march, april and may of year 2010. Leaves finesse expressed by l/L is different: 

march l/L=32÷37%; april l/L=22÷30%; may l/L=16÷28%. Language leaf mature surface is between 71,24÷145,2 

cm². Average mass bulbs = 2,4 g/buc and length by 12 mm to 50 mm. 

Keywords: wild garlic; Allium ursinum L. subsp. ucrainicum; leafes and bulbs; biometry. 

__________________________________________________________________________________________ 


The Allium genus includes approximately 500 

species spread worldwide. Allium ursinum L. is a 

monocots on family Alliaceae and is widely in 

Europa, Asia Minor, Caucasus, Siberia up to the 

Kamchatka Peninsula. 

In Romania this species ”mezohigrofita” grows 

in frequent clusters at the shadows of the trees. It has 

elliptical-lanceolat leaves with white flowers grouped 

and from the biochemical point consist through the 

presence of the ether oils with sulfur, that are giving 

their own smell [1-3]. 

Under various popular names- buckrams, wild 

garlic, broad-leaved garlic, wood garlic, sremuš or 

bear's garlic- this species is used by locals in 

preparations for spring salad and is very appreciated 

for many qualities. 

They have been shown to have applications as 

antimicrobial, antithrombotic, antitumor, 

hypolipidaemic, antiarthritic and hypoglycemic 

agents [4-8]. 

The last researches about the population of A. 

ursinum from Romania put in evidence differences of 

biomass depending of the geographic area and the 

local pedoclimatic conditions. 

Wild garlic is a plant which grows on soils with high 

mineral trophicity and takes place into the 

“megatroph” category with the value V= 85-100 % 

[9]. 

The opportunity of this biometrical and 

phytobiological studies consist in the representation 

of some morphologic-bulbus,folium,flores-by wild 

garlic elements harvested from the Luncavita Forest 

(Macin Mountains), a plant with high pharmaceutic 

potential. 


The harvesting of the bilological material that 

was realized with the agreement of the O.S. Macin 

that manages the area of the Luncavita Forest 

(U.P.I.”Izvorul lui Gavrila”). 


Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) 

The macroscopic exam served as a review of the 

observed characters with free eye or with the 

magnifer and as sensory through the perception of the 

smell and the taste on the informations contained in 

the bibliography of speciality and own researches 

[10, 11]. 

The biometrical observations were obtained 

based on the published biometrical calculation 

methods. The surface of the leaves was measured 

with the help of a mathematic model by the summing 

of the geometrical figures distributed uniformly on an 

sample of 30 leaves [12]. 

Some representative examples indentified on 

the area are stored in the Herbarium of Botanical 

Garden Galati and of the Pharmacy and Medicine 

Faculty of “Dunarea de Jos” University Galati. 


A.ursinum grows on big areas in the Luncavita 

Forest only in north hills or near the water. 

Sometimes this species can be found in other zones 

but in low population. 

The acompaining flora is composed by different 

species like: Corydalis solida, Asarum europaeum, 

Corylus avellana, Tilia tomentosa, Hedera helix, 

Polygonatum latifolium, Scilla bifolia, Carpinus 

betulus, Viola odorata, Ranunculus ficaria, Lamium 

purpureum, Galium aparine, Geum urbanum, 

Anthriscus cerefolium, Muscari botryoides and 

others. 

Our observations show us that under the shrubs 

(Corylus avellana) known for its organic requestsrich 

soils,deep,loose- and a rich litter,wild garlic 

reaches the base of the shrubs [14]. 

Also,the power of growing and penetration of 

the wild garlic was noticed even through the 

woody,halfdescomposed fragments (Fig. 1). 

Simple bulbs or two united can be found at a 

relative depth small in the soil (3-5cm) especially in 

the humus layer and they have good developed roots 

and branched by 3÷15cm length (Fig.2). The 

appearance of the leaves are leveled:first in March, 

the second simultaneous with the third (by case). The 

most of the plants have two leaves. 

68 

Fig. 1. The escape of the wild garlic through 

the wood fragments ( original photo) 

Biometric analysis consists of the following 

items (Table 1): the leaf length (L), the leaf width (l), 

the petiole length (Lp), the percentage ratio-leaf 

finesse (l/L), number ribs (R), mass of green leafs 

(M). 

On the bulbs (Table 2, Fig. 4) were measured 

the length (L), the mass (M), diameter (D) and 

number of the roots (No.roots). 

Our biometric studies made on the leaves of 

A.ursinum shou that the rapport between the width 

and the length of the limb leaf is conversely 

proportional with the procedure of growing (Fig.3):in 

March l/L=32÷37%; in April l/L=22÷30%; in May 

l/L=16÷28%. 

The form of the leaves at the immature plant 

from March is predominant ovat-eliptical and at 

mature in May is elliptical lanceolat. 

The growing in length of the petiole is more 

pronounced in April 104÷280mm. 

The arch parallel nervatiune is numerical 

constant in all of the phases. 

The surface of the limb leaf mature is contained 

between 71,24÷145,2 cm² and the weight of the 

green leaves is 24,49 g/10 mature leaves. 

So it can be confirmed that the foliar biomass 

of the population of A.ursinum from Luncavita Forest 

is lower by comparison with the morphological 

studies on the same harvestes species from Botosani 

area ( 35,85 g/10 leafes) [8].

Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) 

Table 1. Biometrical elements on wild garlic leafes 

Months/ 

Leaf 

number 

m 

a 

r 

c 

h 

a 

p 

r 

i 

l 

m 

a 

y 

L 

mm 

l 

mm 

Lp 

mm 

l/L 

% 

R 

no 

M 

g 

1 145 50 98 34 18 0,71 

2 120 45 81 37 17 0,56 

3 150 50 100 33 19 0,8 

4 110 35 74 32 17 0,57 

5 125 44 81 35 17 0,6 

6 100 35 75 35 17 0,5 

7 148 49 100 33 18 0,75 

8 143 50 100 35 19 0,7 

9 135 45 88 33 17 0,69 

10 149 50 100 33 19 0,79 

1 200 60 256 30 22 1,48 

2 210 60 280 28 22 1,5 

3 157 35 104 22 17 1,04 

4 168 39 106 23 17 1,09 

5 205 59 280 28 22 1,4 

6 195 59 280 30 22 1,35 

7 155 35 105 22 17 1,01 

8 188 57 270 30 22 1,23 

9 190 57 268 30 22 1,26 

10 209 60 280 29 22 1,42 

1 261 70 359 26 23 2,8 

2 185 40 193 22 17 2,31 

3 250 70 360 28 23 2,62 

4 260 71 360 27 23 2,9 

5 240 67 343 27 19 2,38 

6 187 41 192 21 17 2,32 

7 180 39 195 22 17 1,9 

8 259 70 358 27 23 2,79 

9 227 37 324 16 18 2,18 

10 235 38 325 16 18 2,29 

The harvesting of the bulbs was realized in 

February before the entry in vegetation of the plants. 

In the studied area the identified bulbs had different 

sizes (Fig.4): max.length=50mm with the diameter 

D=7mm; min. length =12mm with the diameter 

D=3mm. 

The number of roots is contend between 7÷10. 

The medium mass of the bulb is 2,4 g/piece. 

69 

Fig. 2. Bulbus with radix on wild garlic 

(original photo) 

Table 2. Biometrical elements on 

wild garlic bulbs 

Months/ 

Bulb 

number 

f 

e 

b 

r 

u 

a 

r 

y 

L 

mm 

M 

g 

D 

mm 

No. 

roots 

1 30 3,3 5 8 

2 14 1,2 4 7 

3 25 3,1 5 7 

4 50 4,5 7 10 

5 40 3,8 7 10 

6 13 1,1 3 8 

7 20 1,3 5 7 

8 35 3,6 6 9 

9 12 1,1 3 7 

10 15 1,2 4 7 

Fig. 3. The percentage ratio-leaf finesse wild garlic 

Legend: S1-sample march; S2-sample april;S3sample 

may

Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) 

Fig. 4. Measurements of bulbs on wild garlic 

The infloresecense is umbeliform arranged on a 

florifera strain that passes the height of the leaves. 

The floral stalk leaves from the same place take a 

vaulted form. At the base of the stalks there are 

bacterias wich form an involucres. The flower is type 

3, specific mococotyledonous, and the tricarperal 

ovary crushed emits a specific smell of the garlic and 

has a sweety taste wich attracts the bugs (Fig.5). 

Fig.5. Inflorescence of A. ursinum 

From organoleptic point of view there has been 

seen the next things: all of the vegetal products 

harvested-roots,bulbs,leaves,flowers-they have a 

piquant taste and powerful smell of garlic; the roots 

have second branches and the bulbs (white-yellow) 

are sourrounded by white and transparent 

membranes; the green leaves on the both faces are 

elliptical lanceolat and the pedicels are smooth, that 

means the determination ucrainicum Kleopow et 

Oxner [1,2]. 

70 

The informations from literature of speciality 

about the determination of underspecies of Allium 

ursinum are very little because of the similarities 

between underspecies ursinum and underspecies 

ucrainium. Even, the difference can be realized when 

the plants reach the level of inflorescence. 

With the help of the magnifier can be observed that 

the pedicels don’t prezent papillae and they have a 

smooth surface (Fig.6) characteristic of the 

underspecies ucrainicum [1]. 

Fig.6. “Pediceli” and ovary “tricarpelar” of 

A. ursinum (original photo) 

The spreading of the ursinum underspecies, 

includes areas from Mountains Macin -Greci, 

Tiganca, Niculitel- but there is not specified the area 

of the Luncavita Forest [2]. Also, the recent studies 

realized in North Dobrogea show on the 

Gymnospermio-Celtetum Association the presence of 

the Allium ursinum species but there aren’t any 

references about the underspecies [13]. 


Our studies realized in Luncavita Forest (O.S. 

Macin, U.P. I, “Izvorul lui Gavrila”) shows the 

presence of the wild garlic on large areas but only on 

north hills near water. 

The literature informations of speciality are 

confirmed concerning the exigency of the species 

against trophicity of the soil and our observations 

shows an affinity of the wild garlic by Corylus 

avellana.

Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010) 

We found only an foliar dimorphism in the first 

fenophase in March when the report l/L is high 

32÷37% opposite the values from May l/L=16÷28%. 

The mass of the leaves is 24,49 g/10 the values of the 

leaves is lower comparative with the population of 

the wild garlic from other zones (Botosani) and the 

mature bulbs grow until 50mm length with a mass 

about 2,4g. 

The harvested vegetal products-bulbs,flowershave 

a specific smell of garlic. 

The fitobiological and biological analysis 

permitted us an identify in premiere, of the 

subspecies studied from the Macin Mountains 

(Luncavita Forest) and that would be: Allium ursinum 

L. ssp. ucrainicum Kleopow et Oxner. 

The investigation of the natural population is 

necesarry because of the biosintetical potential 

therefore it can be influented by de pedoclimatic 

conditions from the area of the sampling of the 

plants. 

The studies undertaken by us can offer the 

premise of the harvest,conservation and processing of 

some vegetal products from wild garlic in order to 

improve the farmocognostic researches. 

5. References 

[1] CIOCÂRLAN V., 2000. Flora ilustrată a 

României–Pteridophyta et Spermatophyta, 

Editura Ceres, Bucureşti, pg. 919-925. 

[2] SĂVULESCU T., Flora Republicii Socialiste 

România, Editura Academiei Republicii Socialiste 

România , 1966, Vol. XI, pg.193-266. 

[3] TITA I., 2005. Botanica farmaceutica editia a IIa, 

Editura Didactica si Pedagogica Bucuresti, pg. 

854-863. 

[4 ] DJURDJEVIC L.,, Dinic A., Pavlovic P., 

Mitrovic M., Karadzic B., Tesevic V., 2003. 

Allelopathic potential of Allium ursinum L., 

Biochemical Systematics and Ecology 32, pg.533- 

544. 

[5] ONCEANU (LUPOAE) Mariana, Miron Tudor 

Lucian, Dinica Rodica. Studiul unor principii 

active din specia Alium ursinum recoltată din 

flora spontană, publicat în rezumat, Conferinţa 

Naţională a Societăţii Ecologice din România, 

Galaţi, octombrie 2009. 

71 

[6] STAJNER D., POPOVIC B.M., Canadanovic- 

Brunet J., Stajner M., 2008. Antioxidant and 

scavenger activities of Allium ursinum, 

Fitoterapia 79, p. 303-305. 

[7] ARHANA SENGUPTA et al., 2004. Allium 

Vegetables in Cancer Prevention: An Overview, 

Asian Pacific Journal of Cancer Prevention, Vol 

5: 237-245. 

[8] MIHĂILESCU R., Mitroi G. Iacob E. Miron A., 

Stănescu U., Gille E. , Creţu R., Ionescu E. 

Giurescu C. , 2008. Obtaining of phytoproducts 

for the cardiovascular diseases profilaxy, Note 1 

Some investigation of the Allium ursinum 

chemical composition , The 5’th Conference on 

Medicinal and Aromatic Plants of Southeast 

European Countres , BRNO. 

[9] CONSTANTIN D. CHIRITA et al., 1964. 

Fundamentele naturalistice si metodologice ale 

tipologiei si cartarii stationale forestiere , Editura 

Academiei RPR, pag.110-113 . 

[10] *** FARMACOPEA ROMANA, 1993. Ediţia 

a-X-a , Editura Medicală Bucureşti , pg. 10-63. 

[11] BUCUR L.,ISTUDOR V. et al., 2002. Analiza 

farmacognostica, Instrument de determinare a 

identitatii puritatii si calitatii produselor vegetale, 

Editura Ovidius University Press, Constanta, pg. 

7-87. 

[12] BERCU R., BAVARU A., 2007. Biometrical 

and morpho-anatomical observations on Acer 

monspessulanum L. (Aceraceae) leaves, 

Contributii Botanice, XLII, Gradina Botanica 

“Alexandru Borza” Cluj Napoca, pg. 105-110. 

[13] PETRESCU M. Cercetări privind biodiversitatea 

unor ecosisteme forestiere din Dobrogea de Nord, 

Editura Nereamia, Napocae-Tulcea, pg.61-72, 

2004. 

[14] NEGULESCU E., SAVULESCU Al., 1957. 

Dendrologie, Editura Agro-Silvica de Stat, 

Bucuresti, pg. 184-188.


DINITROPHENYL DERIVATIVES ACTION ON WHEAT GERMINATION 

Cristina Amalia DUMITRAS -HUTANU*, 

*„Al. I. Cuza” University of Iasi, 11 Carol I, 

Iasi-700506, Romania, hutanu_amalia@yahoo.com 

__________________________________________________________________________________________ 

Abstract: Several dinitrophenyl ethers such as 2,4-dinitroanisol, 2,4-dinitrophenetol, 2,4-dinitro-1- 

(octadecyloxy) benzene, 3-(2,4-dinitrophenoxy)propane-1,2-diol or other similar compounds have been 

synthesized and tested comparatively to some well-known metabolic inhibitors and stimulators within the 

germination experiments. As a result, the weight of the resulted plantlets was diminished by 2,4-dinitroanisol and 

3-(2,4-dinitrophenoxy)propane-1,2-diol treatments (1.15 g/lot and 32.03 mg/plantlet in the case of 2,4dinitroanisol; 

0.11 g/lot and 22.3 mg/plantlet in the case of 3-(2,4-dinitrophenoxy)propane-1,2-diol). 

Dinitrophenyl ethers inhibited seed germination, most probably by blocking oxidative phosphorylation. A novel 

mechanism of action of these pesticides was discussed. Consequently,the toxicity processes of these pesticidelike 

compounds and metabolic inhibitors was discussed in direct relationship with their infrared absorbance and 

fluorescence quenching. 

Keywords: pesticide toxicity, dinitrophenyl ethers, dintirophenols, wheat germination. 

__________________________________________________________________________________________ 


Dinitroderivatives, especially the aromatics, are 

frequently used as intermediates in the manufacture 

of pharmaceuticals, dyes, pesticides and explosives. 

They have multiple biological actions, being used as 

insecticides, fungicides, herbicides and acaricides [1, 

2]. However, Environment Protection Agency in 

SUA (EPA) included the dinitrophenols on the list of 

national priorities and in concentration of 3-46 mg 

dinitrophenol/kg body kill; no antidote is known 

(max. admissible dose 70 ppb in water, EPA, 2004). 

It is assumed that dinitrophenols hinder the proton 

translocation through the mitochondrial inner 

membrane and therefore oxidative phosphorylation is 

inhibited (ATP is no longer formed and the cells 

deprive of essential energy supply). It is also possible 

that the dinitrophenols act toxically due to the 

inhibition of formation of some triplet states (instable 

biradicals) by a resonance process with the triplet 

structures in the living cells (A. Szent-Gyorgyi-Nobel 

Prize, 1957) [3, 4, 5, 6, 7, 8]. Because the existing 

data are inconclusive and do not support a precise 

action mechanism of dintrophenyl derivatives on 

living organisms, it was necessary to synthesize some 

dinitrophenols and dinitrophenyl ethers whose 

biological activity should be tested. 

The purpose of this paper is to compare the 

biological activity of some synthetic compounds 

containing the di- and nitrophenyl moiety with that of 

some well-known metabolic inhibitors and 

stimulators. Because germination experiments are 

easy, cheap, fast and spectacular, the testing of the 

action of some action of some known and newly 

synthesized substances on living organisms will be 

performed using germinating cereal seeds [3-5]. The 

possible mechanism of toxicity of these chemicals 

and pesticides are discussed in the light of the 

biostructural theory by Eugen Macovschi as well as 

the chemiosmotic theory by Peter Mitchell [6, 7, 8]. 


Biological material. The wheat samples 

(Triticum aestivum), Henika variety, were taken from 

the Agricultural Research Station in Suceava. The 

1000 seeds weighed 37.2 g and had a residual 

humidity of 12%. Chemical reagents. The reagents 

used were of analytic purity (Merck, Sigma, 

Chimopar) and the solution and the water slurries 

were prepared using redistilled water. Thus, 


Dinitrophenyl derivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) 

dinitroderivatives such as 2,4-dinitrophenetol, 2,4dinitroanisol, 

3-(2,4-dinitrophenoxy)propane-1,2-diol 

and 2,4-dinitrophenyl-glutathione were synthesized. 

Several solutions of dinitrophenyl ethers and 

dinitrophenols with the concentrations 4x10 -3 M were 

prepared. A blank with bidistilled water was also 

carried out. 

Equipment. The chemical syntheses were 

carried out using the organic chemistry lab equipment 

of the Chemistry Department of “Al. I. Cuza” 

University of Iasi. The experiments and the 

germination determinations were performed in Petri 

dishes, on double Watmann no. 1 filter paper at room 

temperature. The separation and purification of the 

compounds obtained were carried out using thin layer 

chromatography on silica gel (Kieselgel 60F254, 

Merck) and on silica gel column. The infrared spectra 

were taken on a Jasco FT/IR660Plus Fourier 

spectrometer in the range from 0 to 15000 cm -1 . 

Procedure. The germination parameters were 

measured according to ISTA recommendations (Seed 

Science and Technology, 1993), however we worked 

also with lots of 50 seeds which were laid to 

germinate on filter paper, in Petri dishes, in three 

repetitions. The first count took place after three days 

(energy of germination, EG), the second after 7 days 

(germination rate, GR). The germinated, abnormal 

and dead seeds as well as the resulting plantlets were 

counted. 

The treatment lasted for an hour, followed by 

the distribution of the seeds uniformly in the Petri 

dishes, on double filter paper, together with the 

treatment solution. The seeds with a visible root were 

considered germinated. The seeds were watered daily 

with 5 ml of redistilled water. The plantlets were cut 

at the level of the seeds 7 days after, measured and 

weighed (height, H, in cm and mass, m, in grams). 

Statistics. The results were processed using the 

Tukey test [9]. The mean square deviation sx of the 

samples was also calculated, as well as t factor, with 

a view to compare the results obtained under the 

action of different treatments. 


As for the stimulative effect, the most active 

substance in these experiments proved to be 

resorcinol, which increased slightly by 5.5% and 

74 

phenylalanine by 6.3% the average mass of plantlets 

as compared to the blank. 2,4-Dinitrophenol inhibited 

total the germination process of wheat seeds, (Table 

1). 

1 2 3 4 5 6 

Fig. 1. The biological effect of some nitrophenyl 

derivatives and other compounds on wheat 

germination. 1 – Blank (water); 2 – DNP; 3 – DNG; 

4 – DNA; 5 – resorcinol; 6 – L-β-phenylalanine. 

Table 1. The toxicity of 2,4-dinitrophenol (DNP), 3- 

(2,4-dinitrophenoxy)propane-1,2-diol (DNG), 2,4dinitroanisol 

(DNA), resorcinol and L-βphenylalanine 

and at concentrations 4x10 -3 M in a 

wheat seeds germination experiment. 

Treatment *) 

1 - Blank 

(water) 

2 - DNP, 

4x10 -3 M 

3 –DNG, 

4x10 -3 M 

4 – DNA, 

4x10 -3 M 

5 – 

resorcinol, 

4x10 -3 M 

Germination 

Rate 

(G. R.) 

Plantlets 

size 

(S, cm) 

Average 

of roots 

mass 

(m, mg) 

90% 6.4+0.6 18.7+1.3 

0% 0 0 

42% 2.5+0.6 8.6+3.2 

85% 4.6+0.6 13.9+0.1 

83% 5.6+0.7 18.9+0.2 

6 – L-βphenylalanine, 

4x10 -3 M 

87% 6.0+0.8 19.1+0.6 

D (Tukey test) 4.3 1.3 1.2 

The height of the plantles treated with 2,4dinitrophenol 

(DNP), 3-(2,4-dinitrophenoxy)propane-1,2-diol 

(DNG), 2,4-dinitroanisol (DNA), 

resorcinol and L-β-phenylalanine and at

Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) 

concentrations 4x10 -3 M, as compared to blank 

(water) treatment. It is apparent from the table that 

the root mass treatments with resorcinol and L-βphenylalanine 

is higher than the control samples. 

Number of 

plantlets 

12 

10 

8 

6 

4 

2 

Blank 

0 

40 60 80 100 120 

Plantlets size (mm) 

Lot A 

Lot B 

Lot C 

Average 

Fig. 2. The height of the plantlets of the lots blanck 

Number of 

platlets 

3 

2 

1 

DNG 

0 

20 30 40 50 60 70 


Lot A 

Lot B 

Lot C 

Average 

Fig. 3 . The height of the plantlets of the lots treated 

with 3-(2,4-dinitrophenoxy)propane-1,2-diol) 

75 

Number of 

plantlets 

10 

8 

6 

4 

2 

DNA 

0 

40 60 80 


Lot A 

Lot B 

Lot C 

Average 

Fig. 4 – The height of the plantlets of the lots treated with 

2,4-dinitroanisol. 

Number of 

plantlets 

10 

8 

6 

4 

2 

Resorcinol 

Lot A 

Lot B 

Lot C 

Average 

0 

40 60 80 100 


Fig. 5. The height of the plantlets of the lots treated 

with resorcinol.

Dinitrophenyl derivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) 

Number of 

plantlets 

Fig. 6. The height of the plantlets of the lots treated 

with L-β-phenylalanine 

Of all the five figures can be seen as only for 

seedlings lengths blank if the three lots are very close. 

In other cases the range of lengths of seedlings was 

higher. These differences show once again disrupting 

the development of seedlings in the presence of 

chemicals, whether stimulatory or inhibitory effect. 

Toxicity 

16 

14 

12 

10 

8 

6 

4 

2 

0 

L-β-phenylalanine 

2.4-Dinitrophenol 

Lot A 

Lot B 

Lot C 

Average 

40 60 80 100 


100 

80 

60 

40 

20 

0 

0 2 4 6 8 10 

Concentration (mM) 

Fig. 7 – The biological effect of 2,4-dinitrophenol on 

wheat germination. 

Toxicity seen in this figure is that of 2,4dinitrophenol 

(DNP). 

76 

At concentrations of 3x10 -3 M, DNP does not 

allow the germination of wheat seeds 

The toxicity mechanism of these pesticides, 

pesticide-like compounds and metabolic inhibitors 

may be discussed in direct relationship to their 

infrared absorbance and fluorescence quenching (not 

shown). Thus, all of them have a significant 

absorbance at about 6000 cm -1 in IR, corresponding 

to ∆G of ATP formation (as previously shown by G. 

Drochioiu, personal communication) and quench the 

fluorescence of tryptophan and other biological 

compounds. Fluorescence quenching of tryptophan 

(1µg/µl) was tested using 2,4-dinitro-ortho-cresol 

(DNOC). The intense quenching activity of DNOC 

was associated with a stronger uncoupling property. 

According to Drochioiu’s hypothesis, one must 

take into consideration the fact that the pH 

modification could be a secondary phenomenon, 

being possible to transfer the energy of triplet states 

to the ADP molecule, which incorporates it as ATP. 

The transition from an excited state to a normal 

state leads to the release or absorption of a proton, 

depending on the acid or base character of the 

compound that is in an excited state. 

2,4-Dinitrophenols act as uncouplers of the 

breathing from oxidative phosphorylation, which 

results in an intensified oxygen consumption, without 

ATP synthesis. Dinitrophenols normally disturb ATP 

production within the cellular mitochondria, because 

the ATP is the molecule which stores and supplies 

energy for cellular activities [6-8]. The present 

theories, such as P. Mitchell’s chemiosmotic theory, 

claim that, unlike the other enzymes in the 

mitochondrial respiratory chain, the ATP pumps 

protons from the intermembrane space towards the 

matrix. Thus, the energy that the other enzymes in the 

chain use to accumulate protons in the intermembrane 

space is recuperated. This energy is necessary for the 

ADP phosphorylation reaction with the mineral 

phosphate, in the presence of Mg ions, the reaction 

being endothermic and requires more than 31 kJ/mol. 

Present research showed that it is possible for 

the dinitrophenyl ethers to act in a non-chemical way, 

probably through radical and triplet status formation, 

and that the proton translocation could be a 

secondary phenomenon in the process of oxidative 

phosphorylation. The action mechanism of the 

compounds investigated is concordant with Eugen

Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010) 

Macovschi’s well-known biostructural theory, but 

contradicts Peter Mitchell’s chemiosmotic 

hypothesis. 


Development of seedlings is disrupted in the 

presence of chemicals, whether stimulatory or 

inhibitory effect. 

Dinitrophenyl ethers and phenolic derivatives 

displayed a similar pattern of biological activity 

inhibiting seed germination, most probably by 

blocking oxidative phosphorylation. Therefore, we 

discussed a mechanism of biological activity as well 

as that of toxicity related to the energy transfer in 

biological systems to form ATP. The proton 

translocation through the biological membranes could 

be a secondary phenomenon, but the most important 

event in the toxicity process of dinitrophenyl 

derivatives. Further research is still necessary to 

clarify the specificity of the biological activity of di- 

and nitrophenols. 

5. References 

[1] BEWLEY J.D., Black M., 1994 - Seeds, 

Physiology of development and germination, 

Plenum press, 2nd Ed., New York and London. 

[2] COMĂRIŢĂ E., Şoldea C., Dumitrescu E., 1986 

- Chimia şi tehnologia pesticidelor, Ed. Tehnică, 

Bucureşti, 188 pp. 

[3] DUMITRAS-HUTANU, C. A., Pui, A., 

Drochioiu, G., 2008 - Dinitrofenil derivati cu 

posibile aplicatii in medicina si biologie: 

mecanisme de actiune si toxicitate, Materiale si 

procese innovative. Simp. V, ZFICPM, Iasi, 

Editura Politehnium, 61-66. 

[4] DUMITRAŞ-HUŢANU C. A., Pui, A., 

Gradinaru, R., and Drochioiu, G., 2008 - 

Toxicity of dinitrophenyl derivatives used as 

pesticides and their environmental impact, 

Lucrări ştiinţifice USAMV Iaşi, seria 

Agricultură, 51. 

[5] DUMITRAŞ-HUŢANU C. A., Pui A., Jurcoane 

S., Rusu E., Drochioiu G. 2009 - Biological 

effect and the toxicity mechanisms of some 

77 

ninitrophenyl ethers. Roum. Biotechnol. Lett. , 

Vol. 14(6), 4893-4899 pp. 

[6] LEHNINGER A. L., 1987 - Biochimie, Ed. 

Tehnică, Bucureşti, Vol. 2, 473, 547 pp. 

[7] DROCHIOIU G., 2006 - In Life and mind. In 

search of the physical basis. S. Savva (ed.) 

Trafford Publ., Canada, USA, Ireland & UK, 43 

pp. 

[8] MITCHELL P., 1978 - David Keilin’s respiratory 

chain concept and its chemiosmotic 

consequences, Nobel Lecture. 

[9] SNEDECOR G. W., 1994 - Statistical methods 

applied to experiments in agriculture and 

biology, The Iowa Stat Univ. Press, 255 pp.


THE ACTION OF SOME INSECTICIDES UPON PHYSIOLOGICAL INDICES IN 

RANA (PELOPHYLAX) RIDIBUNDA 

Alina PĂUNESCU*, Cristina Maria PONEPAL*, Octavian DRĂGHICI*, Alexandru Gabriel MARINESCU* 

* University of Pitesti, Faculty of Science, Departament of Ecology 

Târgu din Vale Street, no.1, Pitesti,410087, Romania, alina_paunescu@yahoo.com 

__________________________________________________________________________________________ 

Abstract: The goal of this work is to study the physiological changes induced by the action of three insecticide 

(Carbetox, Actara 25WG and Reldan 40EC) in Rana (Pelophylax) ridibunda. The animals used in the experiment 

were divided in four experimental lots: two lots of control individuals (first lot was kept at 4-6ºC and the second 

lot at 22-24ºC) and two experimental lots in which the animals were treated with toxic substance and kept at 4- 

6ºC, respectively at 22-24ºC. The toxic was administrated with intraperitoneal shots (one shot every two days, in 

a scheme of three weeks). At the end of the experiment we determinate number of erythrocytes (RBC), leukocytes 

(WBC) and glycemia values. We observe a decrease in number of blood cell (RBC and WBC) as well as an 

increase a glycemia values. 

Keywords: Carbetox, Actara 25WG, Reldan 40EC, frog, erythrocytes, leukocytes, glycemia 

__________________________________________________________________________________________ 


A number of factors have been suggested for 

recently observed amphibian decreases, and one 

potential factor is pesticide exposure. The use of 

pesticides in agriculture can have effects on 

amphibian within or adjacent to application areas [1, 

2]. 

Aside from direct deposition or drift, insecticides 

can reach aquatic habitats via runoff, which depends 

on precipitation, soil conditions, and slope of the 

catchments area [3]. The effect of insecticides on 

large aquatic organisms varies with the test organism. 

Frogs were found to be more sensitive and may serve 

as a biological indicator for pesticide contamination 

in waterways [4]. 

Our goal is to study the effect of three 

insecticides (Carbetox, Actara 25WG and Reldan 

40EC) in some physiological parameters (number of 

erythrocytes and leukocytes, glycemia level) in Rana 

(Pelophylax) ridibunda at two heat level (4-6ºC and 

22-24ºC). 

Carbetox (malathion) is one of the most widely 

used organophosphorous pesticides with numerous 

agricultural and therapeutic applications, and 

exposure to environmentally applied malathion can 

lead to adverse systemic effects in anurans. 

Cutaneous absorption is considered a potentially 

important route of environmental exposure to 

organophosphorous compounds for amphibians, 

especially in aquatic environments [5]. It is slightly 

toxic via the dermal route. 

Actara 25WG is a neonicotinoid insecticide 

active against a broad range of commercially 

important sucking and chewing pests and it has as its 

component the major active ingredient, thiamethoxam 

(25%). Thiamethoxam's chemical structure is slightly 

different than the other neonicotinoid insecticides, 

making it the most water soluble of this family. 

Reldan 40EC is an insecticide from the class of 

organophosphates. The active substance of this 

insecticide is chlorpyrifos. 


Adult specimens of amphibians (Rana 

ridibunda), of both sexes, captured in spring (April- 

May) from the surrounding areas of the city Pitesti 

(Romania) were kept unfed in freshwater aquaria. 

The water was changed daily to avoid the 

accumulation of toxic substances. After 10 days of 

adaptation in the lab, when they were unfed, the frogs 

were separated in lots, which were used separately for 

the following experiments: two lots of control 

individuals, containing animals kept in laboratory at 

4-6 o C, respectively at 22-24ºC with no treatment, in 

ISSN-1453-1267 © 2010 Ovidius University Pres

The action of some insecticides.../ Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) 

running water which was changed everyday, (1) one 

lot containing animals which were subjected to 

treatment with insecticide and kept at 4-6ºC, (2) a 

second lot containing animals which were subjected 

to treatment with insecticide and kept at 22-24ºC. 

The toxic was administered by intraperitoneal 

shots, one shot every two days, in a scheme of 3 

weeks. The administered dosage of insecticide was 

not lethal as none of the subjects died through the 

experiment. We used three different types of 

insecticide: Carbetox (active substance is malathion) 

in a dose of 0.01ml/g body weight, Actara 25WG 

(active substance is thiamethoxame) in a dose of 

0.4mg/g body weight and Reldan 40EC (active 

substance is chloropyrifos-methyl) in a dose of 

0.01ml/g body weight. 

The number of erythrocytes and leukocytes was 

microscopically determined with a Thoma cells 

numbering chamber, by using a small amount of 

blood collected from the heart [6]; the glycemia level 

has been determinate using an Accutrend GCT. 


The number of erythrocytes in the frog 

individuals subjected for three weeks to treatment 

with 0.01ml/g body weight of Carbetox was 

significantly affected as shown in Figure 1. The 

difference between the number of erythrocytes which 

was determined for the control and the ‘treated’ lot at 

4-6ºC, an average decrease of 16.68% was found in 

the treated frog individuals who seemed to be related 

to the intense hemolytic activity. At 22-24ºC we 

registered a decrease with 37.01% in a number of 

erythrocytes. 

In animals treated with Actara 25WG in a dose 

of 0.4mg/g body weight, there has been a decrease in 

the number of erythrocytes with 35.11% to the 

control value for specimens kept at 4-6°C and with 

37.42% for animals treated with insecticide and kept 

at 22-24°C. 

We mention that similar results in the number of 

erythrocytes in the lake frog were obtained by other 

researchers in similar experimental conditions in fish. 

Thus, Ponepal [7] found a decrease in the number of 

erythrocytes in fish under the action of Actara 25WG 

insecticide, as well as a decrease in the oxygen 

consumption. Also, Dhembare [8] recorded decreased 

80 

hemoglobin, the number of erythrocytes, leukocytes 

and platelets in fishes were exposed to LC50 of some 

insecticides for seven days. 

As shown in Figure 1, as compared to the values 

recorded for the control individuals of frog, the 

number of erythrocytes increases by 51.14% for the 

animals which were treated with Reldan 40EC in a 

dose of 0.01 ml/g of body weight and kept at 4-6ºC, 

while animals treated with the same concentration of 

Reldan 40EC but kept at 22-24ºC the number of 

erythrocytes increases by 76.88%. Increased number 

of erythrocytes under the action of Reldan 40EC has 

also been noticed by Păunescu et al [9]. 

number of erythrocytes/ml blood 

1000000 

900000 

800000 

700000 

600000 

500000 

400000 

300000 

200000 

100000 

0 

358166.7 

481111.1 

298400 

225608.2 

232405.6 

301077.8 

851000 

691888.9 

control Carbetox Actara 25WG Reldan 40EC 

Fig. 1. The influence of some insecticide upon 

number of erythrocytes in Rana (Pelophylax) 

ridibunda 

4-6ºC 

22-24ºC 

The number of leukocytes (Fig.2) at the two 

heat levels registered similar changes to that of the 

number of red blood cells as can be seen in Figure 1. 

Carbetox insecticide in a dose of 0.01ml/g body 

weight determined a decrease in number of 

leukocytes with 87.26% as compared with the witness 

value, at 4-6ºC. An intensive leucopenia was also 

registered at 22-24ºC, when the number of leukocytes 

decreases with 101.93%. The number of leukocytes 

decreases by 28.52% to the witness for animals 

treated with Actara 25WG and kept at 4-6°C, while 

the value of this index is lower, 62.06% as compared 

to the witness, at higher temperatures 

(22-24ºC). Reldan 40EC in a dose of 0.01 ml/g of 

body weight was also affected the number of 

leukocytes. As shown in figure 2, the difference 

between the number of leukocytes which was 

determined for the control kept at 4-6ºC and the 

‘treated’ lot kept at the same temperature, an average

Alina Păunescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) 

decrease of 53.07% was found in the treated frog 

individuals. Similar results were obtained at 22-24ºC 

when the numbers of leukocytes decrease by 68.81% 

of the control value. Similar effects have been carried 

out by [10] studying the effects of chloropyrifos on 

mice. 

number of leukocytes/ml blood 

700 

600 

500 

400 

300 

200 

100 

0 

423.9444 

516.3333 

226.3889 209.9444 

303 

195.8889 

198.9444 

161 


4-6ºC 

22-24ºC 


number of leukocytes in Rana (Pelophylax) ridibunda 

The glycemia level was found to be significantly 

influenced by Carbetox insecticide. Thus, as shown in 

Figure 3, at a concentration of 0.01ml/g body weight, 

this index increases after three weeks of treatment to 

61.29% of the control value at 4-6ºC. The same 

concentration of this toxic determinate, at 22-24ºC an 

increase of blood glucose concentration with 

212.09%. It has been reported in several studies that 

hyperglycemia is one of the side effects in poisoning 

by OP in subchronic exposure and in acute treatment 

[11, 12, and 13]. Several studies have demonstrated 

some evidence for damage in pancreatic exocrine 

function after anticholinesterase 

insecticide intoxication [14, 15, 16, and 17]. The 

stimulation of pancreatic secretion secondary to 

cholinergic stimulation seems to be responsible for 

the development of pancreatitis [18, 19, 20, and 21]. 

The influence of Actara 25WG is also felt in the 

glucose level, whose values are shown in Figure 3. Its 

analysis shows an increase of glucose by 85.07% 

compared to witness for animals kept at a temperature 

of 4-6°C and treated with a concentration of 0.4mg/g 

Actara 25WG and 153.05% for the animals kept at 

22-24°C and treated with the same concentration of 

toxic. Reldan 40EC in a concentration of 0.01ml/g 

body weight determinate, after three weeks of 

treatment, an increase of glycemia level with 

127.41% as compared to the witness value in the case 

81 

of animals kept at 4-6ºC and with 173.59% in the 

case of animals kept at 22-24ºC. 

These changes occur due to inhibition of 

glucose tissue by the toxic, and inhibition of Krebs 

cycle and glicolise enzymes, this leading to 

accumulation of glucose in the blood. 

mg glucosis/ml blood 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

11.16667 

22.72222 

18 

34.83333 

20.66667 

57.5 

25.38889 

62.16667 



glycemia in Rana (Pelophylax) ridibunda 


4-6ºC 

22-24ºC 

Analyzing comparatively the influence of three 

insecticides (Carbetox, Actara 25WG and Reldan 

40EC) upon some physiological indices in Rana 

(Pelophylax) ridibunda, we found that these decrease 

(in percentage) the number of erythrocytes and 

leukocytes and increase the glycemia values. Only 

Reldan 40EC insecticide causes an increase in RBC. 

On the other hand, the toxic effect of these 

insecticides was proven to be more powerful at 22- 

24ºC than 4-6ºC. 

5. References 

[1] BERRILL M, BERTRAM S, PAULI B, 1997 - 

Effects of pesticides on amphibian embryos and 

larvae. In: Green DM (ed) Amphibians in decline: 

Canadian studies of a global problem. Reports 

from the declining amphibian population task 

force. Herpetol Conserv, 1:233–245. 

[2] GREULICH K, HOQUE E, PFLUGMACHER S, 

2002 - Uptake, metabolism, and effects on 

detoxication enzymes of isoproturon in spawn and 

tadpoles of amphibians. Environ Toxicol Saf, 52: 

256–266.

The action of some insecticides.../ Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010) 

[3] LIESS M, SCHULZ R, LIESS MHD, ROTHER 

B, KREUZIG R, 1999 - Determination of 

insecticide contamination in agricultural 

headwater streams. Wat Res, 33: 239–247. 

[4] CALUMPANG SMF, MEDINA MJB, TEJADA 

AW, MEDINAJR, 1997 - Toxicity of 

Chlorpyrifos, Fenubucarb, Monocrotophos, and 

Methyl Parathion to fish and frogs after a 

Simulated Overflow of Paddy Water. Bull. 

Environ. Contam. Toxicol., 58: 909-914. 

[5] WILLENS S, STOSKOPF M, BAYNES R, 

LEWBART G, TAYLOR S, KENNEDY- 

STOSKOPF S, 2006 - Percutaneous malathion 

absorption by anuran skin in flow-through 

diffusion cells. Envtl. Toxicol. & Pharm, 22: 

263-267. 

[6] PICOŞ CA, NĂSTĂSESCU GH, 1988 - Lucrări 

practice de fiziologie animală. Tipografia 

Universităţii din Bucureşti, Bucureşti, 107, 122- 

123, 192-195. 

[7] PONEPAL MC, PĂUNESCU A, DRĂGHICI O, 

MARINESCU AlG, 2006 - Research on the 

changes of some physiological parameters in 

several fish species under the action of the 

thiametoxame insecticide. In: Proceedings 36th 

International Conference of IAD: 163-167. 

[8] DHEMBARE AJ, PONDHA GM, 2000 - 

Hematological changes in fish, Punctius sophore 

exposed to some insecticides. Journal 

Experimental Zoo India, 3(1): 41-44. 

[9] PĂUNESCU A, PONEPAL CM, DRĂGHICI O, 

MARINESCU AlG, 2009 - The influence of 

Reldan 40EC insecticide upon physiological 

indices in Rana ridibunda. Lucrări Ştiinţifice 

USAMVB Seria B, LIII: 173-178. 

[10] AMBALI S, AKANBI D, IGBOKWE N, 

SHITTU M, KAWU M, AYO J, 2007 - 

Evaluation of subchronic chlorpyrifos poisoning 

on hematological and serum biochemical changes 

in mice and protective effect of vitamin C. The 

Journal of Toxicological Sciences, 32: 111-120. 

[11] GUPTA PK, 1974 - Malathion induced 

biochemical changes in rats. Acta Pharmacol. 

Toxicol., 35(3): 191–194. 

[12] RODRIGUES MR, PUGA FR, CHENKER E, 

MAZANTI MT, 1986 - Short term effect of 

malathion on rats’ blood glucose and on glucose 

82 

utilization by mammalian cells in vitro. 

Ectotoxicol. Environ. Safety, 12 (2): 110–113. 

[13] MATIN MA, HUSAIN K, 1987 - Cerebral 

glycogenolysis and glycolysis in malathiontreated 

hyperglycaemic animals. Biochem. 

Pharmacol., 36(11): 1815–1817. 

[14] GOKEL Y, GULALP B, ACIKALIN A, 2002 - 

Parotitis due to organophosphate intoxication. J. 

Toxicol. Clin. Toxicol. J., 40(5): 563–565. 

[15] PANIERI E, KRIGE JE, BORNMAN PC, 

LINTON DM, 1997 - Severe necrotizing 

pancreatitis caused by organophosphate 

poisoning. J.Clin. Gastrenterol., 25: 463–465. 

[16] DRESSEL TD, GOODALE RL, ARNESON 

MA, BORNER JW, 1979 - Pancreatitis as a 

complication of anticholinesterase insecticide 

intoxication. Ann. Surg., 189: 199–204. 

[17] LANKISCH PG, MULLER CH, 

NIEDERSTADT H, BRAND A, 1990 - Painless 

acute pancreatitis subsequent to anticholinesterase 

insecticide (parathion) intoxication. Am. J. 

Gastroenterol., 85: 872–875. 

[18] KANDALAFT K, LIU S, MANIVEL C, 

BORNER JW, DRESSEL TD, SUTHERLAND 

DE, GOODALE RL, 1991 - Organophosphate 

increases the sensitivity of human exocrine 

pancreas to acetulcholine. Pancreas, 6: 398–403. 

[19] GOODALE RL, MANIVEL JC, BORNER JW, 

LIU S, JUDGE J, LI C, TANAKA T, 1993 - 

Organophosphate sensitizes the human pancreas 

to acinar cell injury: an ultrastructural study. 

Pancreas, 8: 171–175. 

[20] WEIZMAN Z, SOFER S, 1992 - Acute 

pancreatitis in children with anticholinesterase 

insecticide intoxication. Pediatrics, 90: 204–206. 

[21] MORITZ F, DROY JM, DUTHEIL G, MELKI 

J, BONMARCHAND G, LEROY J, 1994 - Acute 

pancreatitis after carbamate insecticide 

intoxication. Intens. Care Med., 20: 49–50.


CHANGES OF SOME PHYSIOLOGICAL PARAMETERS IN PRUSSIAN CARP 

UNDER THE ACTION OF SOME FUNGICIDE 

Maria Cristina PONEPAL, * Alina PĂUNESCU*, Alexandru Gabriel MARINESCU*, Octavian DRĂGHICI* 

* Universitatea din Piteşti, Facultatea de Ştiinţe 

Str. Tg. din Vale, nr.1 Piteşti, România, e-mail: ponepal_maria@yahoo.com 

__________________________________________________________________________________________ 

Abstract: This study was carried out to analyze the effects of sublethal and lethal concentrations of Bravo 500 

SC, Champion 50 WP, Tilt 250 and Tiradin 70 PUS fungicide on some physiological parameters (oxygen 

consumption, breathing frequency, number of erythrocytes) of the prussian carp (Carassius auratus gibelio 

Bloch). The acute and subacute toxicity of fungicides was evaluated in glass aquaria under semi-static conditions. 

Keywords: prussian carp, fungicide, Bravo, Champion, Tilt, Tiradin, breathings frequency, oxygen consumption 

__________________________________________________________________________________________ 


The commercial product Bravo 500 SC is a 

concentrated suspension of chlorothalonil (500g / l); 

chlorothalonil (2,4,5,6 tetrachlor isophthal-nitrile) is 

a contact fungicide with curative and preventive 

action (works by stopping germination and the 

development of spores) for combating a large number 

of pathogens (leaf spots, downy mildews, 

alternarioses, fruit rots, brown tor of fruit, scab) that 

threaten the main crops [1]. The fungicide is part of 

group IV of toxicity; it is not toxic to bees, warmblooded 

animals and moderately toxic to insects [2]. 

Chlorothalonil and its metabolites are very toxic to 

fish, aquatic invertebrates and marine organisms [3]: 

LC50 (96 h) is of 0.25 mg/l for rainbow trout (Salmo 

gairdneri), 0.3 mg/l for sun perch (Lepomis 

macrochirus), 0.43 mg/l for sea devil (Ictalurus 

punctatus), etc. 

Champion WP (copper hidroxide) is a fixed 

copper fungicide widely used for control of fungal 

and bacterial pathogens. Copper is highly toxic in 

aquatic environments and has effects in fish, 

invertebrates, and amphibians, with all three groups 

equally sensitive to chronic toxicity [4]. The 

Champion WP product is toxic to fish and aquatic 

organisms (96-hour LC50 Bluegill: 180 mg/l, 96-hour 

LC50 Rainbow trout: 0.023 mg/l and 48-hour EC50 

Daphnia: 0.065 mg/l). 

Tilt 250 (the active ingredient is propiconazole 

– triazole fungicide) has protective, curative and 

systemic activity. Propiconazole's mode of action is 

demethylation of C-14 during ergosterol biosynthesis, 

and leading to accumulation of C-14 methyl sterols. 

The biosynthesis of these ergosterols is critical to the 

formation of cell walls of fungi [5]. The 

propiconazole is non toxic for bees, invertebrates and 

soil bacteriae, but is dangerous for fish and ather 

aquatic organisms (LC50 values ppm for freshwater 

fish species: bluegill 1.3-10.2, brown trout 3.5, 

rainbow trout 0.9-13.2, carp 6.8-21.0, catfish 2.0-5.1 

and fathead minnow 7.6) [6] , [7]. 

Tiradin fungicide (the active substance is the 

thiuram - tetramethylthiuram disulphide TMTD) is a 

general use contact fungicide with protective action, 

third group of toxicity. Dithiocarbamates form a large 

group of chemicals that have numerous uses in 

agriculture and medicine [8]. It is used to control 

Botrytis on fruit and vegetables and in seed 

treatment. The 96-hour EC50 for algae growth 

inhibition is approximately 1 mg/l (1 ppm), the 48hour 

EC50 for Daphnia is less than 0.21 ppm and the 

96-hour LC50 for fish is approximately 0.1 ppm 

(Bluegill sunfish, 0.0445 mg/l, Rainbow trout, 0.128 

mg/l and 4 mg/l carp) [9]. 

This study was carried out to analyze the effects 

of sublethal and lethal concentrations – of some 

fungicide: Bravo 500 SC (from 0.078125 x 10 -3 to 

12.5 x 10 -3 ml/l water), Champion 50 WP (from 

0.003 to 3 mg/l water), Tilt 250 (from 0.25 to 4 ml/l 

water) and Tiradin 70 PUS (from 0.01 to 0.16 ml/l 


Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) 

water) on some physiological parameters (oxygen 

consumption, breathing frequency, number of 

erythrocytes 0.078125 x 10 -3 and 1.5625 x 10 -3 ml 

Bravo/l water, 0.003 mg Champion/l water and 1 ml 

Tiradin/l water) of the prussian carp (Carassius 

auratus gibelio Bloch). 

The acute and subacute toxicity of this 

fungicide was evaluated in glass aquaria under semistatic 

conditions. 


Determinations were made between January 

2004 and October 2009 on prussian carp samples 

(Carassius auratus gibelio Bloch), captured from the 

surrounding rivers of Piteşti. Animals were 

acclimatized for 10 days before the completion of 

experiments in aquariums with a capacity of 100 l 

and 50 l [10], under conditions of natural 

photoperiodism, a period in which they were fed once 

a day (ad libitum), at around 10 am. 

After acclimatization in the laboratory, the fish 

were separated in two experimental variants (lots of 

10-20 fish - average weight 18 g) subjected to 

fungicides. 

I. Determinations of oxygen consumption and 

frequency of respiratory movements at intervals of 

24, 48, 72, 96, 168 and 336 hours on all samples of 

these lots (depending on survival) on prussian carp 

subjected to: 

- I.1. Bravo 500 SC in concentrations of 

0.00078125, 0.0015625, 0.003125, 0.0625, 0.0125 

ml /l water and the control lot 

- I.2. Champion 50 WP in concentrations of 

0.003, 0.03, 0.3 and 3 mg/l water and the control lot 

Tilt 250 in concentrations of 0.25, 0.5, 1, 2, 4 ml /l 

water and the control lot 

- I.3. Tiradin 70 PUS in concentrations of 0.01, 

0.02, 0.04, 0.08, 0.16 ml /l water and the control lot 

II. Hematological determinations (after one, 

respectively two weeks of exposure to the fungicide, 

the fishs were sacrificed to achieve intakes of blood 

necessary to hematological calculations (number of 

erythrocytes). 

84 

II.1 – fish subjected to Bravo 500 SC in 

concentrations of 0.00078125, 0.0015625, ml /l 

water and the control lot 

II.2 – fish subjected to Champion 50 WP in 

concentrations of 0.003 mg/l water and the control lot 

II.3 - fish subjected to Tilt 250 in concentration 

1ml /l water and the control lot 

The fungicides concentrations were determined 

by preliminary tests of survival. The introduction of 

fish in solutions was done after their mixing and 

aeration for 5 minutes. The water temperature was 

16-18°C, the "immersion" solution was changed 

every 24 hours, and aeration of water was continuous; 

the fish were not fed during experiments to avoid 

further intervention of this factor [10]. The testing 

method was systematic with refreshing solution at 24 

hours after the calculations of the day, in aquariums 

of 100 l (50 l, respectively) for each experimental lot. 

Determination of oxygen consumption was 

done by means of the oximetre and Winkler method 

and erythrocytes were counted with Thoma chamber, 

using a small amount of blood from the caudal artery 

on the optic microscope [10], [11]. The statistical 

interpretation of the results was performed with 

ANOVA (LSD) test. 


The first four figures (fig.1-4) shows the 

average frequency of the respiratory movements of 

prussian carps exposed to the action of some 

fungicide (Bravo, Champion, Tilt and Tiradin).

Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) 

Fig.1. The influence of Bravo fungicide upon 

breathing frequency on prussian carp 

Fig.2. The influence of Champion fungicide upon 


Fig.3. The influence of Tilt fungicide upon 


85 

Fig.4. The influence of Tiradin fungicide upon 


Bravo and Champion have changed the 

respiratory rhythm of prussian carps in all 

investigated concentrations. For all concentrations 

tested the effect of the fungicide is initially 

stimulating and inhibitory as regards the frequency of 

respiratory movements. In two experimental variants 

(0.01 and 0.02 ml/l water)Tiradin is stimulating of the 

breathing frequency of fish; at the concentration of 

0.04, 0.08 and 0.16 ml/l water, the fungicide caused 

a decrease in the respiratory rhythm of prussian carps. 

Changes of prussian carps oxygen consumption 

exposed to the action of Bravo, Champion, Tilt and 

Tiradin fungicides in differrent concentrations are 

shown in fig. 5-8. 

Fig.5. The influence of the Bravo fungicide upon 

oxygen consumption on prussian carp


Fig.6. The influence of the Champion fungicide 

upon oxygen consumption on prussian carp 

Fig.7. The influence of the Tilt fungicide upon 

oxygen consumption on prussian carp 

Fig.8. The influence of the Tiradin fungicide 

upon oxygen consumption on prussian carp 

86 

Clinical symptoms observed during fungicide 

exposure (Bravo, Champion, Tilt and Tiradin) of 

prussian carp, correspond to observations by other 

authors reporting on the toxicity of fungicides [12], 

[13], [14]. 

Common symptoms of initial acute exposure to 

fungicides have apparent fish hypoxia, disoriented 

(ataxic) at the surface, and mucus-producing effects. 

The oxygen consumption was found to be 

significantly influenced by the concentration of the 

used fungicides. 

Bravo 500 SC ,in concentrations of 0.78125 x 

10 -3 , 1.5625 x 10 -3 , 3.125 x 10 -3 , 6.25 x 10 -3 and 12,5 

x 10 -3 ml / l Bravo, had an overall stimulating effect 

on oxygen consumption of prussian carps in the first 

phase (with variable duration: 24-96 hours after 

exposure) followed by restoration of energy 

metabolism after 7 days of exposure to toxic. Tiradin 

and Tilt have an inhibitory effect on the energy 

metabolism of prussian carps. After 7 days of 

exposure to Tilt, for all lots of fish tested, oxygen 

consumption values fall below the value recorded 

before the introduction of fish in experiments. 

Decreased oxygen consumption under the action 

of some pesticides and changes in respiratory rate 

(Dithane M 45, Reldan, Tilt,) has also been noticed 

by Marinescu [12] and Ponepal [13], [14]. 

Figure 9 show the changes in the average values 

of erythrocytes after one and two weeks of exposure 

to some fungicides. 

Fig. 9. The influence of some fungicide upon 

number of erythrocytes on prussian carp

Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010) 

Champion 0.003 1 1 1 9 9 9 

After 7 and 14 days of exposure to three 

0 0 0 

fungicide (Bravo, Champion and Tiradin) we found 

0.03 1 1 1 9 8 8 

out a significant decrease in the number of 

0 0 0 

erythrocytes. Similarly results were obtained in carp 

0.3 1 1 1 9 8 7 

by Hughes [15] after a brief exposure to 

0 0 0 

Methadathion. The decrease in RBC after 7 days 

3 1 1 9 8 6 4 

exposure to some pesticides in fish was observed by 

0 0 

Dhembare and Pondha [16], Ponepal et al. [13], 

Contr 1 1 1 1 10 9 

[14]. 

ol lot 0 0 0 0 

The fungicide Tilt, in concentration of 1 ml/l Tilt 0.25 1 1 1 1 10 10 

water has an stimulatory effect of erythocytes 

0 0 0 0 

number. 

0.5 1 1 1 1 9 9 

In experimental variants with Tiradin and Tilt 

0 0 0 0 

have only been observed three stages of the 

1 1 1 1 9 9 8 

sympthomatologicycal scheme described by 

0 0 0 

Schäperclaus for the intoxicated fish [10]. 

2 8 6 3 2 0 0 

Neurotoxic effects in rats from thiram exposure 

4 7 4 0 0 0 0 

has been noticed by Lee and Peters [17]. 

Table 1 shows the data on fish mortality during 

the experiments. 

Chlorothalonil toxicity is lower than that 

indicated in the literature [2], [3], which is due both 

to the testing method (semi-static) and the fact that no 

pure chemical product has been used. 

Tiradin 

Contr 

ol lot 

0.01 

0.02 

0.04 

0.08 

0.16 

1 

0 

10 

10 

10 

10 

8 

1 

0 

10 

10 

10 

10 

7 

1 

0 

10 

10 

10 

8 

6 

1 

0 

10 

10 

10 

8 

4 

10 

10 

10 

8 

6 

1 

9 

10 

10 

6 

5 

1 

Contr 10 10 10 10 10 10 

Table 1. Lethal effect of some fungicide on 

prussian carp 

ol lot 

Experimental 

variants 

(fungicide 

Conc 

entrat 

ion - 

ml/l, 

mg/l 

Bravo 0.000 

7812 

5 

0.001 

5625 

0.003 

125 

0.006 

25 

0.012 

25 

Contr 

ol lot 

The number of living 

specimens 

Immersion time (hours) 

24 48 72 96 168 33 

6 

10 10 10 10 10 10 

10 10 10 10 9 9 

10 10 9 9 8 7 

10 10 9 9 8 6 

10 10 9 8 7 2 

10 10 10 10 10 10 

87 


The fungicides investigated (Bravo, Champion, 

Tilt and Tiradin) have changed the respiratory rhythm 

of prussian carps. For all concentrations tested the 

effect of Bravo and Tilt fungicide is initially 

stimulating and inhibitory as regards the frequency of 

respiratory movements. In two experimental variants 

(0.01 and 0.02 ml/l water) Tiradin is stimulating of 

the breathing frequency of fish; at the concentration 

of 0.04, 0.08 and 0.16 ml/l water, the fungicide 

caused a decrease in the respiratory rhythm of 

prussian carps. 

The fungicide Bravo, had an overall stimulating 

effect on oxygen consumption of prussian carps in the 

first phase followed by restoration of energy 

metabolism after 7 days of exposure to toxic. 

The fungicide Champion, under the 

concentrations of 0.003 and 3 mg/l water, had, after


96 hours of exposure, a stimulatory effect on oxygen 

consumption for the prussian carp. 

The other two fungicides tested (Tiradin and Tilt) 

have an inhibitory effect on oxygen consumption for 

the prussian carps. 

After seven and 14 days of exposure to Bravo 

500 SC (0.078125 x 10 -3 to 12.5 x 10 -3 ml/l water) 

and Champion (0,003 mg/l water) at 16-18 ºC we 

found out a significant decrease in the number of 

erythrocytes of prusian carp. Tilt 250, in 

concentration of 1 ml/l water causess a increase in the 

prussian carps erythrocytes (after 7 and 14 days of 

exposure). 

5. References 

[1] http://extoxnet.orst.edu/pips/chloroth.htm 

[2] KIDD H and JAMES DR, 1991 - Eds. The 

Agrochemicals Handbook, Third Edition. Royal 

Society of Chemistry Information Services, 

Cambridge, UK, (as updated). 6-10 

[3] DAVIES PE AND WHITE RWG, 1985 - The 

toxicology and metabolism of chlorothalonil in 

fish. 1. Lethal levels for Salmo gairdneri, Galaxias 

maculatus, G. truttaceus and G. auratus and the 

fate of super(14)C-TCIN in S. gairdneri , Aquatic 

Toxicology, 7 (1-2). pp. 93-105. 

[4] HORNE MT and DUNSON WA, 1995 - Effects 

of low pH, metals, and water hardness on larval 

amphibians, Archives of Environmental 

Contamination and Toxicology, 29:500-505 

[5] THOMSON WT, 1997- Agricultural Chemicals. 

Book IV: Fungicides. 12th edition, Thomson 

Publications, Fresno, CA 

[6] http://www.epa.gov/ngispgm3/iris/irisdat 

[7] http://www3.bae.ncsu.edu/info1/courses 

[8] HOWARD PH, 1989 - Pesticides. In : Handbook 

of Environmental Fate and Exposure Data for 

Organic Chemicals, Lewis Publishers, Chelsea, 

MI, pp.4-20 

[9] www.epa.gov/HPV/pubs/summaries 

[10] PICOS CA, NASTASESCU GH, 1988 - Lucrări 

practice de fiziologie animală. Tipografia 

Universităţii din Bucureşti, p.107, 122-123, 192- 

195. 

[11] ŞERBAN M, CIMPEANU G, IONESCU 

EMANUELA, 1993 - Metode de laborator în 

88 

biochimia animală, Editura Didactică şi 

Pedagogică, Bucureşti, 252 pp. 

[12] MARINESCU AG, DRĂGHICI O, PONEPAL 

C, PĂUNESCU A, 2004 - The influence of 

fungicide (Dithane M-45) on some physiological 

indices in the prussian carp (Carassius auratus 

gibelio Bloch), International Association for 

Danube Research, Novi Sad, 35: 209-214 

[13] PONEPAL MC, PĂUNESCU A, MARINESCU 

AG., DRĂGHICI O, 2009 - Effect of the 

Fungicide Chlorothalonil (Bravo) on Some 

Physiological Parameters in Prussian Carp, 

Lucrări ştiinţifice USAMV Iaşi, seria 

Horticultură, vol 52. 

[14] PONEPAL M., PĂUNESCU A, MARINESCU 

AG, DRĂGHICI O, 2009 - The Changes of Some 

Physiological Parameters in Prussian Carp Under 

The Action of the Tilt Fungicide, Bulletin 

UASVM, Cluj, 2009, 66. 

[15] HUGHES G., SZEGLETES T, NEMCSOK KJ. 

1995 - Haematological and biological changes in 

the blood of carp (Cyprinus carpio) following 

brief exposure to an organophosphoric insecticide 

(Methidathion),Abs.Int.Biond.Symp.Cesze 

Budejovice, May 

[16] DHEMBARE AJ, PONDHA GM, 2000 - 

Haematological changes in fish. Punctius sophore 

exposed to some insecticides, J.Expt. Zoo. India, 

3(1), 41-44. 

[17] LEE CC and PETERS PJ, 1967 - Neurotoxicity 

and behaviour effects of thiuram in rats, . Envir. 

Health Perspectives, 17:35-43.


CYTOGENETIC EFFECTS INDUCED BY MANGANESE AND LEAD 

MICROELEMENTS ON GERMINATION AT TRITICUM AESTIVUM L. 

Elena DOROFTEI 1 , Maria Mihaela ANTOFIE 2 , Daciana SAVA 1 , Marioara TRANDAFIRESCU 1 

1 Faculty of Natural and Agricultural Science, „Ovidius” University, Constantza, University Street No. 1, Bilding 

B, Campus, 900552, Romania, email: edoroftei2000@yahoo.ca 

2 Faculty of Agricultural Sciences, Food Industry and Nature Potection, University “Lucian Blaga”from Sibiu 

__________________________________________________________________________________________ 

Abstract: Our study is about the effects of manganese and lead microelements treatment on germination at 

Triticum aestivum L. The cytogenetic effects were studied by the calculation of the mitotic index, by the study of 

the interphase and chromosomal aberrations on the mitotic cells. We used MnSO4 and Pb(NO3)2 solutions with 

different concentrations: 0.0001, 0.005, and 0.01%. The Triticum seeds were preliminary imbued in water, and 

then they were treated for 6 and 24 hours in these solutions. The control group was treated with water. We 

prepared five cytological slides, for each slide we have studied 10 microscopic fields with good density of cells 

for the mitotic index and another 10 different microscopic fields for abnormal interphases and chromosomal 

aberrations. In the analyzed meristematic cells we observed an almost totally inhibition of cell division and the 

mitotic index was smaller in comparison with the control variant. The study of the frequency of the cells in 

different phases of the mitotic division showed that the highest percent was registered by prophases, followed at 

distance by telophases. We can conclude that the heavy metals Mn and Pb have a significant mutagenic activity 

in vivo upon the radicles of Triticum aestivum L. 

Keywords: Triticum aestivum, cytogenetic effects, lead, manganese, mitotic index, chromosomal aberrations. 

__________________________________________________________________________________________ 


Aside pesticides - the most important „stress 

indicators” which are especially used in agriculture, 

other very important indicators are heavy metals. 

The residual waters resulting from the galvanic 

industry contain a real “hurricane” of heavy metals 

such as: mercury, cadmium, zinc, copper, lead and 

chrome. Generally the water pollution sources for 

heavy metals are as following: galvanic industry, 

mining, metallurgy and car industry. Copper water 

pollution is especially due to viticulture as the 

copper sulphate is used for pests’ control. 

Lead is eliminated mostly as a result of 

burning gasoline, petrol and different dyes, 

affecting the central nervous system in humans, 

creating behaviour problems and convulsions, at 

higher levels being lethal. Lead is spearing no 

organ or system being the first incriminated in 

boosting or getting worse a series of diseases 

through diminishing the body resistance. Lead 

effects are usually irreversible. 

Manganese is a nutritionally essential 

chemical element but also in certain conditions it 

can be potentially toxic. Manganese name is 

originating from Greek language meaning “magic” 

and this feature is still adequate because the 

scientists are still working to understand different 

effects of its deficiency and toxicity effects for 

living organisms. However, without doubt in high 

levels manganese is highly toxic causing a series of 

pathologies based on reactive oxygen species 

(ROS) generation. 

Long term oxidative stress consequences in 

human where associated to the different diseases 

pathogenesis and toxicities namely atherosclerosis, 

diabetes, chronically inflammatory diseases, 

neurological disturbances and cardiovascudiseases. 

Manganese induces the oxidative stress in a 

time and concentration depending manner, 

according to the cytotoxic parameters 

measurements, lactate dehydrogenase and lipid 

peroxidation. Also, manganese may accumulate 

into the cell causing cytotoxic effects and cell 


Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) 

destruction. Following different activity enzyme 

alteration and the alteration of gene expression the 

intracellular disruptions caused by manganese 

include DNA helix broken up, chromosomes 

destruction and lipid peroxidation (Brooks, 1994). 

Our research focused in detecting the 

mutagenic effects induced by heavy metals such as 

manganese (Mn) and lead (Pb) on higher plants 

using the cytogenetic analysis in Triticum aestivum 

L. as plant indicator for heavy metals polluting 

degree in crops. 

The toxicity symptoms induced by heavy 

metals in plants are the results of some negative 

effects on physiological processes including: 

respiration and photosynthesis inhibition, water – 

plant relationship disruption, decreasing 

plasmalema permeability in root cells, adverse 

effects on the metabolic enzymes (Arduini, 1994; 

Chardonneres et al., 1999; Ouzounidou, 1994; 

Vangronsveld and Clijsters, 1994; Vennitt and 

Parry, 1984). 

2. Materials and Methods 

The chemical effects on chromosomes are 

often studied on plant material such as root tips as 

they are easily produced through seed germination, 

the experiments may be conducted all over the year 

and are not costly (Bateman,1977). 

For studying the heavy metal effect on mitosis 

we used solutions of MnSO4 and Pb(NO3)2 in 

different concentrations (0,0001%; 0,005% and 

0,01%) in which were submersed Triticum seeds 

for 6 and 24 hours, in Petri disches. As control it 

was used tap water. Fragments of young roots were 

fixed into a mixture solution of ethylic alcohol and 

glacial acetic acid in a volumetric rapport of 3:1 for 

16 h in refrigerator followed by a gentle acidic 

hydrolysis in HCl 1N solution for 5 min at 60°C. 

The roots are coloured through the Feulgen method 

using the Schiff reactive for 90 min followed by a 

water bath for 20 min. The slides were prepared 

applying squashing method and the samples were 

analyzed in light microscopy for the cytogenetic 

effects of heavy metals by calculating the mitotic 

index and revealing the chromosomal aberrations 

for different mitotic stages (Doroftei et al., 2008). A 

90 

Novex Holland digit camera was used for taking 

photographs. 

Table 1. Heavy metal concentrations and durations 

used for Triticum aestivum seeds treatments 

Heavy 

metal 

MnSO4 

MnSO4 

Pb(NO3)2 

Pb(NO3)2 

Concentration Variant 

name 

0,0001% V1 

0,005% V2 

0,01% V3 

0,0001% 

0,005% 

0,01% 

0,0001% 

0,005% 

0,01% 

0,0001% 

0,005% 

0,01% 

In this study 5 slides per variant were analyzed 

and for each slide 10 microscopically filed were 

used for mitotix index calculation and for 

chromosomal aberrations study. 


V4 

V5 

V6 

V7 

V8 

V9 

V10 

V11 

V12 

Treatment 

duration 

6 hours 

24 hours 

6 hours 

24 hours 

Analyzing the control untreated roots it was 

revealed the normal feature of the chromosomes 

and also normal cell division behaviour with a 

mitotic index of 15 %. Roots development was 

lower when the Triticum seeds were immersed into 

the tested solutions, macroscopically differences 

being observed compared to the control. Thus, the 

treated roots were smaller and in a low number 

compared to the control. 

The mitotic index significantly decreased 

especially in the case of 0.01% and 24 h treatment 

duration for manganese and lead too, supporting the 

idea that cell division is slower progressing 

compared to the control (Tab. 2). These 

microscopically observations are supported by 

those macroscopically (root number and size). 

The chromosomal aberrations are relatively 

diverse, being aleatory distributed and depending

Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010) 

on manganese and lead concentration and treatment 

period. 

For a treatment with MnSO4 solution in 

concentration of 0.01% for 24 h treatment, it was 

observed cells with big nuclei and unorganized and 

vacuolated features. The un-organizing process is 

due probably to some disequilibrium occurred as a 

consequence of genetic material accumulation in a 

too big quantity. The treatment with MnSO4 

solution in concentration of 0.01% for 6 h treatment 

it was observed that the majority of cells were in 

interphase and prophase and after 24 h of treatment 

cell plasmolysis occurred for non dividing cells. 

The treatment with Pb(NO3)2 solution in 

concentration of 0,0001% and 0,005%, for 6 or 24 

h treatments, induced a decrease in mitotic division 

frequency. For a treatment with Pb(NO3)2 of 

0,01%, for 24 h a significant decrease cells in 

mitotic division frequency it was registered as a 

result of summing the effects of high concentration 

and long period of treatment. For this later variant, 

nuclei unregulated in shape and size were observed 

and chromosome appeared either big with a relaxed 

chromatin either small but presenting a compact 

chromatin and unregulated shape. For lead too, for 

a concentration of 0.01% for 6 h there were 

observed predominantly cells in interphase or 

prophase and after 24 h of treatment the cell 

plasmolysis occurred in the non-dividing cells. 

The studied heavy metals solutions may have 

according to our results the following negative 

effects: 

1. slowing down the cell division rate (figs. 

1-16) 

2. frequent cell degradation appearance (figs. 

7,8,9,10,11,13,14 ) 

3. dehydration effect at cell level frequently 

inducing cell plasmolysis, more drastically 

at 24 h treatment (figs. 9,10,11,15,16,17) 

4. heterochromatinization during prophase 

(figs. 6,7,8,13,14) 

5. changes in the nuclei shape becoming 

elongated (figs. 6,7,8) 

91 

6. degradation of the nucleic material in the 

completed destroyed cells (figs. 

6,7,8,13,14) 

7. an early chloroplasts formatting can be 

observed (figs. 9,10,12,15,16,17). 

In all variants, comparing to the control, a 

decreasing in the mitotic index was observed (figs. 

1, 2, 3, 4). We recorded the lack of cells in 

anaphase for the following variants: 3, 6 and 11. 

For the control as well as for the treated variants the 

predominance of prophase and telophases towards 

the metaphases and anaphases was registered (fig. 

5). 

The highest percentage of dividing cells is 

registered for the variant no. 1 (4%) and it is shown 

in tab. no. 2. The biggest number of cells in 

prophase (19) in variant 1 was registered (tab. 2). 

For metaphase the biggest number of cells was 

registered in variant 7 (14) followed by in variants 

1 and 9 (11). 

These data support the idea that among heavy 

metals, manganese in large quantities impedes the 

normal roots growth for higher plants as a 

consequence of cell division negative effects 

induced for the meristematic cells in Triticum 

aestivum. 

Faze de diviziune % 

6 

5 

4 

3 

2 

1 

0 

Profaze 

Metafaze 

Anafaze 

Telofaze 

Fig. 1. Mitotic division phase’s frequency in 

Triticum aestivum treated with MnSO4 for 6 hours.



6 

5 

4 

3 

2 

1 

0 

Fig. 2. Mitotic division phase’s frequency in 

Triticum aestivum treated with MnSO4 for 24 

hours. 


Fig.3. Mitotic division phase’s frequency in 

Triticum aestivum treated with Pb(NO3)2 for 6 

hours. 


6 

5 

4 

3 

2 

1 

0 

6 

5 

4 

3 

2 

1 

0 

M t V i t V i t V i t 

Fig.4. Mitotic division phase’s frequency in 

Triticum aestivum treated with Pb(NO3)2 for 24 

hours. 

Profaze 

Metafaze 

Anafaze 

Telofaze 

Profaze 

Metafaze 

Anafaze 

Telofaze 

Profaze 

Metafaze 

Anafaze 

Telofaze 

92 

Fig.5. Root meristematic cells in control of 

Triticum aestivum. It can be observed cells in 

prophase, metaphase, anaphase and telophase and 

cytochinesis (200x). 

Fig.6. Root meristematic cells in Triticum aestivum 

treated with MnSO4 0.0001% for 6 h. It can be 

observed cells without content and cells in prophase 

and telophase. The nuclei are hypertrophic with 

obviously hetero-chromatinisations and 

vacuolisations (150x).




observed cells without content and cells in prophase 

and cytochinesis. The nuclei are hypertrophic with 

obviously hetero-chromatinisations and 

vacuolisations (150x). 



observed plasmolytic cells mixed with cells in 

prophase and telophase abnormal disposed and 

containing hypertrophic nuclei with obvious heterochromatinisations 

(150X). 

93 



observed abnormal disposed cells without content 

mixed with plasmolytic cells in witch we can 

observed an early chloroplasts formatting (600X). 

Fig.10. Root meristematic cells in Triticum 

aestivum treated with MnSO4 0.0001% for 24 h. It 

can be observed long cells disposed in lines; cells 

are plasmolytic, during prophase and in witch we 

can observed an early chloroplasts formatting 

(600X).



aestivum treated with MnSO4 0.005 % for 24 h. It 


are plasmolytic, during prophase (600X). 


aestivum treated with MnSO4 0.01% for 24 h. It 


are strongly plasmolytic, during prophase and in 

witch we can observed an early chloroplasts 

formatting (600X). 

94 


aestivum treated with Pb(NO3)2 0.0001 % for 6 h. 

It can be observed cells without content alternating 

with cells in prophase and telophase, having nuclei 

hypertrophic (400X). 


aestivum treated with Pb(NO3)2 0.005 % for 6 h. It 

can be observed cells without content alternating 

with cells in prophase and telophase, having nuclei 

hypertrophic (400X).






can observed an early chloroplasts formatting 

(600X). 


aestivum treated with Pb(NO3)2 0.005 % for 24 h. 

It can be observed long cells disposed in lines; cells 


can observed an early chloroplasts formatting and 

curly cell’ s walls (600X). 

95 





can observed an early chloroplasts formatting and 

curly cell’ s walls (600X). 


Based on the results of this study we may 

conclude that: 

The heavy metals solutions used in this 

experiment have a great mutagenic effect on the 

root meristematic cells of Triticum aestivum 

After the heavy metals solution treatment a 

decrease in cell division in rate was recorded 

The heavy metals have a dehydration effect at 

cellular level 

In all variants a decrease in the mitotic index 

compared to the control was observed 

The mutagenic effects depends on the used 

heavy metals in the treatment and the treatment 

duration 

In the treated cells an early chloroplasts 

formatting can be observed. 

Cytogenetic tests on Triticum aestivum reveal a 

decrease in mitotic index after the treatment with 

the heavy metals solutions. These results revealed 

that the studied heavy metals present a significant


mutagenic activity. The inhibition of mitotic 

division in the root apex induces the root growth 

inhibition as an active reaction of the plant when 

plants are exposed to the action of heavy metals in 

soil. Heavy metal effects are more profound but 

they may become visible using further molecular 

techniques. 

These results are sufficient serious arguments 

in the elaboration of prophylactic methods for 

pollution combating of surface land water, 

underground water as well as for grounding the 

protection measures for ecosystem maintaining. 

5. References 

[1] BROOKS, R.R., 1994: Plants and 

hyperaccumulate heavy metals. In: Plant and 

chemical elements, 88-105. Edited by M.E. 

Farago. Ed. VCH, Weinheim, New York, Basel, 

Cambridge, Tokio. 

[2] ARDUINI, I., GOLDBOLD, D.L., ONNIS, A., 

1994: Cadmium and cooper change root growth 

and morphology at Pinus pinea and Pinus 

pinaster seedling. Physiol. Plant., 92, 675-680. 

[3] CHARDONNERES, A.N., BOOKUM, W.M., 

VELLINGE, S., SCHAT, H., VERKLEIJ, 

J.A.C., ERNST, W.H.O., 1999: Allocation 

patterns of zinc and cadmium in heavy metal 

96 

tolerant and sensitive Silene vulgaris. J. Plant. 

Physiol., 155(6), 778-787. 

[4] OUZOUNIDOU, G., 1994: Cooper induces 

changes on growth, metal content and 

photosynthetic function of Alysum montaneum 

L. plants, Environ. Exp. Bot., 34, (2), 165-172. 

[5] VANGRONSVELD, J., CLIJSTERS, H., 1994: 

Toxic effects of metals. In: Plants and the 

chemical elements, 150-177. Edited by M.E. 

Farago. Ed. VCH, Weinheim, New York, Basel, 

Cambridge, Tokio. 

[6] VENNITT, S., PARRY, J.M., 1984: 

Mutagenicity testing: a practical approach. Ed. 

IRL Press, Oxford, Washington DC. 

[7] BATEMAN, A.J., 1977: Handbook of 

mutagenicity - Test procedures. Edited by B.J. 

Kilbey, M. Legator, W. Nichols, C. Ramel. Ed. 

Elsevier, North Holland, Amsterdam. 

[8] DOROFTEI, E., MIRON, L., ROTARU- 

STĂNCIC, M., 2008: Efectul mutagen al 

metalelor grele cupru şi cadmiu la Allium cepa 

L. (The mutagenic effect of heavy metals 

cooper and cadmium at Allium cepa L.) În: 

Ardelean, A., Crăciun, C. (eds), Analele 

Societãţii Naţionale de Biologie Celularã, XIII, 

225-229, Risoprint, Cluj-Napoca.


Table 2. Number of analysed cells for citogenetic studies regarding the effects of heavy metals 

manganese and lead on cell division 

Variant Total 

studied 

cell 

Martor 

Total 

interphase 

cells 

Total 

division 

cells 

Total 

prophase cells 

97 

Total 

metaphase 

cells 

Total 

anaphase 

cells 

Total 

telophase 

cells 

Nr. Nr. % Nr. % Nr. % Nr. % Nr. % Nr. % 

1000 850 85 150 15 51 5,1 40 4,0 30 3,0 29 2,9 

V1 1000 960 96 40 4,0 19 1,9 11 1,1 6 0,6 4 0,4 

V2 1000 968 96,8 32 3,2 14 1,4 6 0,6 4 0,4 8 0,8 

V3 1000 981 98,1 19 1,9 9 0,9 4 0,4 - - 6 0,6 

V4 1000 980 98 20 2,0 10 1,0 5 0,5 4 0,4 1 0,1 

V5 1000 976 97,6 24 2,4 11 1,1 6 0,6 3 0,3 4 0,4 

V6 1000 975 97,5 25 2,5 12 1,2 9 0,9 - - 4 0,4 

V7 1000 966 96,6 34 3,4 9 0,9 14 1,4 5 0,5 6 0,6 

V8 1000 965 96,5 35 3,5 15 1,5 9 0,9 6 0,6 5 0,5 

V9 1000 966 96,6 34 3,4 12 1,2 11 1,1 8 0,8 3 0,3 

V10 1000 974 97,4 26 2,6 11 1,1 4 0,4 3 0,3 8 0,8 

V11 1000 979 97,9 2,1 2,1 12 1,2 6 0,6 - - 3 0,3 

V12 1000 979 97,9 2,1 2,1 11 1,1 4 0,4 1 0,1 5 0,5


PROBLEMS OF THE HARMONIZING ENVIRONMENTAL 

LEGISLATION AT THE COMPARTMENT "PISCES" IN THE 

REPUBLIC OF MOLDOVA 

Petru COCIRTA, Olesea GLIGA 

Institute of Ecology and Geography (Academy of Sciences of Moldova). 

Academy Str. No.1, Chișinău, MD-2028, Republica Moldova 

E-mail: pcocirta@hotmail.com, camiprim@inbox.ru 

__________________________________________________________________________________________ 

Abstract: In the paper are presented some results regarding principal characteristics on the structure, qualitative 

and comparative analysis of the national acts with EU directives as well with EU and ISO standards. It was 

demonstrated the compatibility of some national legislation and normative acts with EU ones. Special attention 

was dedicated to the rare and endangered species of fish. It was created databases on environmental legislativenormative 

acts of the Republic of Moldova at the compartment “Fishes”, which shows a various and satisfactory 

number of acts in this domain. In the final part of the paper are presented some conclusions and proposals on the 

development of legislation and norms regarding fish species in the Republic of Moldova in accordance with EU 

and international requirements. 

Keywords: fishes and environmental legislation and normative acts state. 

__________________________________________________________________________________________ 


According to the Declaration of Rio de Janeiro 

in 1992 and Agenda 21 [1], protection of biological 

diversity is one of the global environmental 

problems, which depends on addressing the quality of 

life and existence of the living organism son the 

earth. 

Development and conservation of the diversity 

of ichthyofauna species are of paramount importance 

in the management of biological diversity in the 

marsh and aquatic ecosystems in the Republic of 

Moldova [2]. 

In the past 100 years anthropogenic pressure on 

aquatic and march ecosystems has changed cardinal 

the quantity and quality of aquatic biological 

diversity. In Republic of Moldova the aquatic and 

marsh (water areas of rivers, lakes, dam lakes, ponds) 

ecosystems were limited to 94,6 thousand ha (2.8% 

of total territory), and are unevenly distributed and 

characterized by a wide variety of ecological, 

physical, geographical, hydrochemical, 

hydrobiological etc. particularities. Hydrographical 

network consists of three main rivers - the Danube, 

Dniester and Prut, as well as of 3260 rivulets and 

3532 lakes. Most of rives were damaged, destroyed 

or channeled. Biodiversity includes 160 flora and 

125 fauna (vertebrates) species. Hydrofauna recorded 

over 2135 species, including ichthyofauna - 82 

species [3-5]. 

In recent decades the influence of anthropogenic 

factors (industrial pollution, eutrophication 

progressive, toxicity, reducing water flow, etc.) upon 

ecosystems river Dniester and Prut, small rivers in the 

territory of the country makes major changes in 

biodiversity of the hydro-biocenozes with loses the 

viability and biological significance of rivers into the 

biosphere and environment. 

As a consequence, fish resources, which are one 

of the important indicators statuses of the aquatic 

ecosystems, decreased sharply in majority natural 

water objects of the Republic of Moldova and a 

number of species (sterlet, barbel, zarte and others) 

are endangered. In the Red Book of the Republic of 

Moldova (Second edition, 2001) [6] it was included 


Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) 

12 species of fishes (14,6 % of total number). 

Acording to investigations made by Usatai [7], it 

takes place the process of replacement of valuable 

species with less valuable. 

Political and socio-economic reforms in 

Moldova ware conditioning the need to change of 

attitudes towards use of natural resources, promoting 

economic and social development compatible with 

the environment. After the 2009 parliamentary 

elections was conditioned the need to promote the 

new ideas and actions for to harmonize relations in 

the system “Man-Society-Nature”. In this context in 

the Republic of Moldova there are implemented the 

National Strategy „Agenda 21” [8] and a number of 

existing national programs: The Moldovan Village, 

2005-2015; Program of the stabilization and 

economic recovery of the Republic of Moldova for 

the years 2009-2011 [5], and new one: Rethink 

Moldova. Priorities for Medium Term Development, 

2010-2013 [10], which would help “de facto” to 

economic development through solving the 

environmental problems and respectively to stop the 

pollution of the environmental and degradation their 

components. 

Current state of water areas of the Republic of 

Moldova induces new provocations on the 

elaboration of measures to develop the actions and 

current species diversity of ichthyofauna, the 

improvement, utilization and sustainable conservation 

of hydrofauna in general. 

In the program „Rethink Moldova. Priorities 

Medium Term Development” among other priority 

issues that require to be solving there are the 

approximation of legislation and normative acts to 

those of the European Union. Achieving these 

desiderates requires needs updating of the existing 

legislative-normative base, elaboration new laws and 

regulations and/or modification of those existing, 

adaptation national standards and normative to those 

international ones and/or takeover of international 

standards of the ISO and EN Series, etc. 

This desiderates will fully covers the domain 

“Ichthyofauna”, including the section “Fishery”. 

A comprehensive study should be carried out in full 

for the evaluation of legal-normative basis and for 

highlighting some perspective problems for to legal 

100 

ensuring of the environmental management in 

biodiversity conservation domain. 

In this work are presented analytical 

information on current level of legislative-normative 

assurance of the environmental management of 

ichthyofauna species diversity in the Republic of 

Moldova and on forming of the base of legislativenormative 

acts in this domain. 

The aim of work: 

- analyze of legal and normativ systems on 

compartment "Fishes" of UN, EU and Republic of 

Moldova; 

- assessment and completing the data base of 

the acts referred to the Republic of Moldova; 

Highlighting the problems of the legislativenormative 

development in the Republic of Moldova 

on ichthyofauna domain; 

- elaboration of the proposals for harmonization 

of legislation and normative in the domain of 

ichthyofauna to the Strategy of Sustainable 

Development of the Republic of Moldova, to the 

respective EU and international acts; 

In this work are presented analytical and 

summary information on the current level of 

insurance protection activities of ichthyofauna in the 

Republic of Moldova and creation of the base of 

legislative-normative acts in referred domain. 


Study of information regarding legislativenormative 

acts was performed through analyse of the 

data banks, catalogs and other official materials of 

the international and national environmental 

organizations. 

Collecting of acts materials was effectuated in 

the frame of the official publications (written or 

electronic forms) of Secretariats of the international 

conventions, International Organization for 

Standardization, European Union and others, as well 

as from the Republic of Moldova - periodical 

publication "Monitorul Oficial a Republicii 

Moldova", Websites of the Parliament, Government, 

Ministry of Justice and Ministry of Environment and 

others. 

Given the fact that information accumulated in 

ihthiofauna domain will serve to comparative analysis 

of national acts to those international, especially to

Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010) 

European ones and will be used to develop concrete 

recommendations on the harmonization of legislation, 

normative and standards, it was take into account the 

respective international methodological 

recommendations [11-18] and the those of national 

order - Standards of the Republic of Moldova on the 

principles and methodology of standardization (SM 

1-0:2003, MS 1-12:2002; SM 1-20:2002, SM 1- 

21:2002 [18, 19]) and others. 

Storage of the specialized information and 

creation of databases of legislative-normative acts on 

biodiversity domein (EU Directives, International 

Conventions, National legislation and normative) was 

made in electronic form. 

Collecting and processing of information on 

standards and technical regulations was effectuated by 

using existing databases of international and national 

Websites and formation of a register of operative 

information in this domain. 


In the Program "Rethink Moldova. Medium 

Term Development Priorities, 2010-2013” 

approximation of legislative and normative acts to 

those of the EU is among the priority issues, that need 

resolving operational. Collection and analysis of 

material under mentioned program has permitted to 

highlight the following aspects of the assessment and 

the need of International, European and National acts 

for the ecological management on "Fish" 

compartment in the Republic of Moldova. 

3.1. Assessment and training normative legislative 

base in "PISCES" 

3.1.1. International legislation 

Various multilateral environmental agreements 

or conventions have been concluded for nature 

protection in general, and for aquatic fauna in special. 

The European Community takes an active part in the 

elaboration, ratification and implementation of 

multilateral environmental agreements. Republic of 

Moldova also is a part of those conventions and made 

different action in accordance with ratified 

conventions. The principal of them which cover also 

the Fish compartment are named chronologically 

below (see Box 1). 

101 

Box 1: Multilateral environmental agreements 

on nature protection 

• Convention on Wetlands of International 

Importance Especially as Waterfowl 

Habitat (Ramsar, 1971) 

• Convention on International Trade in 

Endangered Species of Wild Fauna and 

Flora (Washington, 

• 1973) 

• Convention on Conservation of Migratory 

Species of Wild Animals (Bonn, 1979) 

• Convention on the Conservation of 

European Wildlife and Natural Habitats 

(Bern, 1979) 

• Convention on Biological Diversity (Rio de 

Janeiro, 1992) 

• Convention on Cooperation for the 

Protection and Sustainable Use of the 

Danube River (Sofia, 1994) 

3.1.2. EU legislation 

In accordance with EU recommendations [10- 

14], were taken to record the majority of legislative 

and normative acts, which are part of the acquis of 

Environment and need to be transposed into national 

law. Environmental acquis recommended for 

harmonization of national legislation is considerably 

smaller (118 documents). As the compartment “Fish” 

there was highlighted the following: 

EU Fish Protection Legislation. Within this 

framework, EU Nature conservation policy is 

implemented by one main piece of legislation – 

Habitats Directive - the Council Directive 

92/43/EEC of 21 May 1992 on the conservation of 

natural habitats and of wild fauna and flora. The 

Directive aim to provide protection for listed species 

and habitats and to create the European ‘coherent 

European ecological network of sites – called Natura 

2000 to enable the maintenance or restoration of 

natural habitat types and the habitats of species at 

favorable conservation status (Art. 3, Habitats 

Directive). The Habitats Directive requires Special 

Areas of Conservation (SACs) to be designated for 

listed plant and animal species, and habitats. 

Together, SACs and Special Protection Areas 

(SPAs) from Birds Directive (Council Directive 

79/409/EEC of 2 April 1979 on the conservation of 

wild birds) make up the Natura 2000 sites. SPAs and


SACs areas can overlap. The Natura 2000 network 

already comprises more than 20,000 sites, covering 

almost a fifth of the EU territory. 

Besides this directive there are further relevant pieces 

of EU nature protection legislation referred to fish, 

summarized in Box 2. 

Box 2: EU nature (fish) protection related 

legislation 

• Council Directive 92/43/EEC on the 

conservation of natural habitats and of wild 

fauna and flora 

• Council Directive 1999/22/EC relating to the 

keeping of wild animals in zoos 

• Council Regulation (EC) No. 338/97 on the 

protection of species of wild fauna and flora 

by regulating trade therein 

Other EU legislation relevant to nature (fish) 

protection include: 

• Environmental Impact Assessment Directive 

(85/337/EEC), amended by Council Directive 

97/11/EC, 

• Access to Environmental Information 

Directive (90/313/EEC), 

• Reporting Directive (91/692/EEC). 

3.1.3 Legislative-normative acts of the Republic of 

Moldova. 

On June 1, 2010 database of legislativenormative 

acts of Republic of Moldova in the 

ichthyofauna domain and interdependent ones 

represents an impressive set of legal materials, 

namely: 

• 6 international environmental conventions to 

which Moldova is party; 

• 13 Laws of the Republic of Moldova; 

• 1 Presidential Decree of Republic of Moldova; 

• 44 acts of the Republic Moldova subordinate to 

laws, from wich 7 Decisions of Parliament, 35 

Decisions of Government, 2 Acts of the Central 

Environmental Authority; 

• 1 Concept; • 2 Strategies; • 2 State Programs. 

The above mentioned has highlighted the 

importance of databases in this domain and the need 

to maintain and develop them. Analyse of the results 

obtained show that the development of legislations 

102 

and normative in the ichthyofauna domain is 

satisfactory. 

Political and social reforms from recent years 

have highlighted the need to harmonize legislativenormative 

acts, inclusive the ichthyofauna domain, to 

the international requirements, which will allow 

fulfilling the obligations of the Republic of Moldova 

Government, assumed by signing the international 

environmental conventions and facilitating the 

process of integration in European Union. In this 

context, there is evident the tendency and efforts for 

significant changes of legislative-normative acts of 

the Republic of Moldova, which started in last 5-6 

years by applying the mechanism of their 

harmonizing to requirements of the international 

legislation and normative, and, in particular, to the 

European requirements, in accordance with 

international obligations of the country. 

However, we should mention that several 

legislative-normative acts from Republic of Moldova 

have prescriptive nature and contain general 

provisions that regulate, primarily, the relations of 

animal kingdom protection and conservation, the 

management of the state protected natural areas and 

others. There is poorly developed legislative base for 

protection of natural complexes, for creating a green 

housing (frame) and application of stringent measures 

for recovery of environmental condition, which have 

directly impacts the habitats of fish species, a special 

vulnerable species. 

In national legislation lacks the mechanism 

needed to optimal ensuring of the protection and 

conservation activities of natural habitats of many 

species of fish, as well as of communities of the 

aquatic plant and animals. 

3.1.4 Legislative issues on the conservation of rare 

and vulnerable species 

Republic of Moldova legislation covers the 

most part of the activities of rational use and 

conservation of ichthyofauna species (Law on Animal 

kingdom (1995), Law on State Protected Areas Fund 

(1998), Law on fund of fisheries, fisheries and fish 

culture (2006), Red book of Moldova (2001). 

Special attention is devoted to rare and 

vulnerable fish species that are protected by several 

legislative acts, the main ones being: the Law on 

State Protected Areas Fund, which includes 15


species, the Red Book of Moldova - 12 species. We 

also note the primary importance of the Berne 

Convention (1979) to which Moldova is part from the 

year 1993. 

Harmonize national legislation with 

international and European requirements impose 

additional measures to conserve species of 

ichthyofauna. Were subjected to comparative analysis 

some legislative acts of Republic of Moldova and 

those more important international (European Red 

List (2009) [21], Council Directive 92/43/EEC [22] 

and the Berne Convention [23] regarding the status 

and the protection state of rare and vulnerable fish 

species. It were analyzed the status of 21 important 

species of fish presents on territory of the Republic of 

Moldova (Table 1). 

Table 1. Some important fish species of the Republic 

of Moldova under comparative analysis 

Name of Species Acts with 

species found 

Order Salmoniformes 

1. Hucho-hucho (L) – Danube 

salmon or Huchen 

2. Salmo salar (L) - 

Atlantic salmon 

3. Umbra krameri(Walbaum) – 

European Mudminnow 

LAK, LPA, RB, 

ERL, CD, BC 1) 

ERL, CD, BC 

LAK, LPA, RB, BC 

Order Cipriniformes 

4. Rutilus frisii Nordmann – LAK, LPA, RB, 

Black Sea Roach 

ERL, CD, BC 

5. Leuciscus leuciscus (L) – 

Common Dace 

LAK, LPA, ERL 

6. Leuciscus idus (L) 

LAK, LPA, RB, 

- Ide or Golden Orfe 

ERL, BC 

7. Vimba-vimba (L) – Zarte LPA, ERL 

8. Barbus barbus borysthenicus LAK, LPA, RB, 

(Dubowsky) – Borys 

ERL 

9. Barbus meridionalis (Petenyi LAK, LPA, RB, 

Heckel) – Mediterranien Barbel ERL, CD, BC 

10.Cobitis taenia (L) – Spined 

Loach 

CD, BC 

103 

11.Gobio albipinnatus 

CD, BC 

(Vladykov Fang) – White-finned 

Gudgeon 

12.Rhodeus sericeus amarus CD, BC 

(Bloch) – European Bitterling 

Order Gadiformes 

13.Lota lota (L) - Barbot 

LAK, LPA, RB, 

ERL 

Order Perciformes 

14.Zingel zingel (L) – Zingel LAK, LPA, RB, 

ERL, CD, BC 

15.Zingel streber (Siebold) - LAK, LPA, RB, 

Sreber 

ERL, CD, BC 

Order Acipenseriformes 

16.Gimnocephalus schraetzer 

(L) - Schraetzer 

17.Huso huso (L) - 

European Sturgeon 

18.Acipenser guldenstaedti 

colchilus (V.Marti) – Russian 

Sturgeon 

19.Acipenser stellatus (Pallas) - 

Sturry Sturgeon 

20.Acipenster ruthenus (L) - 

Sterlet 

21.Acipenster nudiventris 

(Lovetyki) – Bastard Sturgeon 

CD, BC 

LAK, LPA, RB, 

ERL, CD, BC 

LAK, LPA, RB, CD 

LAK, LPA, RB, CD, 

BC 

LPA, CD, BC 

CD 

Note: 1) LAK - Law on Animal Kingdom, LPA - Law on 

State Protected Areas Fund, RB - Red book of Moldova , 

ERL - European Red List, CD - Council Drective 

92/43/EEC, BC - Bern Convention. 

Analysis demonstrates that of these above 

mentioned, only six species (Danube Salmon, Black 

Sea Roach, Mediterranean Barbel, Zingel, Streber, 

European Sturgeon) are covered by all legislation 

acts under review, 12 species are covered by the 

European Red List, 15 species - Council Directive 

92/43/EEC and 15 species - Bern Convention, 

respectively. 

There were identified 9 species, which in 

accordance with requirements of Council Drective 

92/43/EEC falling under Annex II and requires the 

designation of special areas of conservation, as wel as 

under the Bern Convention, ratified by the Moldovan 

Parliament decision No. 1546-XII of 23. 06. 93. But 

5 of these species (Salmo salar, Cobitis taenia, 

Gobio albipinnatus, Rhodeus sericeus amarus,


Gimnocephalus schraetser) have no-one protected 

status in the Republic of Moldova. Also were 

identified 2 species (Acipenster rutenus and 

Acipenster nudiventri) falling under Annex V of the 

Council Directive 92/43/EEC, but the second species 

has no one of any protected status in country. 

The comparative analysis demonstrates the 

need to review the rarity status of the mentioned fish 

species and/or their inclusion in the Red Book of 

Moldova, in other legislation acts and/or performing 

other actions to perpetuate their best. These findings 

are in full compliance with existing legal basis of the 

Republic of Moldova: Art. 9, 16, 17 and 18 of the 

Law on the Red Book of Moldova (No. 325-XVI 

from 15. 12. 2005); The Common Action Plan 

Republic of Moldova – European Union, 2005-2007; 

and The Program "Rethink of Moldova. Priorities for 

medium term development". 

Comparative analysis of the state and how to 

protect rare and vulnerable species of fish confirmed 

the importance of measures taken and existing needs 

in Moldova in this section. Increasing vulnerability a 

ichthyofauna species confirmed by increasing number 

of introduced species in the Red Book of Moldova, 

the second edition of. Proposals have already been 

developed [7] introducing other four species in the 

next edition (III) of the Red Book. They are supposed 

to be: Tench - Tinca tinca (L), Spirlin - Alburnoides 

bipunctatus rossicus (Berg), Crucian Carp - 

Carassius Carassius (L.), Wels catfish - Silurus 

glanis (L.). 

3.2. Evaluation and formation standards database 

in „PISCES” 

In the compartment "Pisces", relative to other 

domains there are few sets of standards, gained 

worldwide by international organizations - ISO 

(International Standardisation Organisation) and IEC 

(International Electrotechnical Commission) and at 

the European level - CEN (European Committee for 

Standardization), CENELEC (European 

Electrotechnical Committee for Standardization) and 

ETSI (European Telecommunications Standards 

Institute) [17.18]. 

3.2.1. International standards 

In accordance with electronic information 

provided by the Organization ISO [17], in querying 

104 

for section "Fish" can be found 17 ISO standards and 

one in elaboration and for section "Fishing and fish 

breeding" another 13 ISO standards. Meanwhile at 

the European level [18] for the section "Fish" it was 

highlighted 12 EN standards, from which 8 ISO 

standards taken by EN (European Normatives) 

organization. 

3.2.2. Standards of the Republic of Moldova 

The Republic of Moldova doesn’t have in action 

ISO and EN standards. But in Catalogue of normative 

documents in standardization of the Republic of 

Moldova [19.20] in 65.150 section “Fishing and fish 

farming” were not identified standards in use, and in 

section 67.120.30 “Fish and fish products” are in 

force 109 GOST standards (standards of Russia 

adopted as national). It is clear the need of some 

decisions and activities in developing regulations and 

standards in the relevant field. 


Republic of Moldova dispose a significant base 

of legislative-normative acts in ichthyofauna domain. 

The legal regulation of ichthyofauna conservation is 

in continuous development, already having a solid 

theoretical and practical basis. 

In the last 4-5 years it is obvious trend and 

efforts for significant changing of the legislativenormative 

acts of the Republic of Moldova by 

applying of the mechanism to their harmonizing to 

the requirements of international legislation and 

normative, especially, to the European requirements. 

In connection with the increasing vulnerability 

of species further efforts are needed for continuous 

development of legislative basis regarding the 

habitats protection of vulnerable species of fish and 

protection of natural complexes in general, as well as 

creation a ecological housing and application of 

stringent measures to redress the environmental 

status. 

Comparative analysis of the state and protection 

mode of rare and vulnerable fish species has 

confirmed the importance of measures taken and 

existing needs in the Republic of Moldova in this 

domain. 

Additional measures are needed on improving 

the mechanism and instruments to ensure optimal


operation of the protection and conservation of many 

species of fish natural habitats, as well as of plant and 

animal aquatic communities. 

In Republic of Moldova the ichthyofauna 

domain needs to move quickly to adopt international 

and European standards in national practice. 

Given that the diversity of fish species in 

Moldova is in its own way, unique and, under current 

conditions of climate change, utilization and damage 

of the ichthyofauna species genetic fund, there is 

need more attention, a stricter approach and effective 

activities for resolution of their development and 

conservation problems. 

5. References 

[1] Agenda 21, Rio de Janeiro, 1992. 

[2] Republic of Moldova. Biological Diversity 

Conservation National Strategy and Action Plan. 

(Ministry of the Environment and Territorial 

Development. The World Bank), Chişinău, 

Ştiinţa, 2002, 100 p. 

[3] Republic of Moldova, First National Report on 

Biological Diversity. (Ministry of the 

Environment and Territorial Development. The 

World Bank), Chişinău, Ştiinţa, 2000, 68 p. 

[4] Republic of Moldova. Third National Report on 

the implementation of the Convention on 

Biological Diversity. CBD, Chisinau, December, 

2005. 

[5]http://bsapm.moldnet.md/Text/Raportul%20III/Ra 

pr-03-englez.pdf - data of access 4 June 2010 

Republic of Moldova. State of the environment 

Report 2006. Ministry of Ecology and Natural 

Resources, Chişinău, 2007, 85 p. 

[6] Red Book of the Republic of Moldova, Second 

edition, Stiinta, 2001, 288p. 

[7] USATÂI M. “Evolution, conservation, and 

sustainable use of diversity of ichthyofauna in 

aquatic ecosystems of Republic of Moldova”. 

Autoreferat of dissertation for the scientific 

degree of doctor Habilitatus in biological 

sciences. Chişinău, 2004, 48 p. (In Romanian) 

[8] National Strategy of the Sustainable Development 

– “Moldova 21”. Supreme Economic Council 

under President of the Republic of Moldova, 

PNUD Moldova, Chişinău, 2000, 129 pag. (In 

Romanian). 

105 

[9] National program “Moldovian Village, 2005- 

2015; Program for stabilization and re-launch of 

the economy in the Republic of Moldova for years 

2009-2011 (In Romanian) - www.gov.md. 

[10] Government of Moldova. Rethink Moldova. 

Priorities for Medium Term Development. Report 

for the Consultative Group Meeting in Brussels 

24 March 2010 

http://siteresources.worldbank.org/INTMOLDOV 

A/Resources/Rethink-Moldova-2010-2013-Finaledit-110310.pdf 

[11] White Paper on the Preparation of the 

Associated Countries of Central and Eastern 

Europe for Integration into the Internal Market of 

the Union, COM(95) 163 final, 3.5.1995 

[12] Environmental regulatory reform in the NIS: the 

case of the Water sector. Twelfth meeting of the 

EAP Task Force, 18-19 October 2000, Almaty. 

http://www.oecd.org/dataoecd/23/5/2382097.pdf 

[13] Guide to the approximation of the European 

Union Environmental Legislation, SEC (97) 1608 

of 25.08.1997. 

http://ec.europa.eu/environment/guide/contents.ht 

m 

[14] Handbook on the implementation of ec 

environmental legislation. 

http://ec.europa.eu/environment/enlarg/handbook/ 

handbook.htm 

[15] COCIRTA P., CLIPA Carolina. Ecological 

legislation of the Republic of Moldova: Catalogue 

of the documents. Chisinau, Stiinta, 2008, 65 pag. 

(In Romanian) 

[16] COCIRTA P. Environmental systems and 

electronic information’s in the Republic of 

Moldova. Academy of Sciences of Moldova. 

Institute of Ecology and Geography – Chisinau, 

2007. 30 pag. (In Romanian) 

[17]http://www.standardsinfo.net/info/livelink/fetch/2 

000/148478/6301438/index.html 

18. http://www.cen.eu/cen/pages/default.aspx 

[19] Catalogue of normative documents in 

standardization of the Republic of Moldova. Year 

2008. National Institute of Standardization and 

Metrology. Vol.1,2,3. Chisinau, 2008. (In 

Romanian) 

[20] http://www.standard.md; 

[21] European Red List. - www.iucnredlist/Europe 

[22]http://ec.europa.eu/environment/nature/legislatio 

n/habitatsdirective/index_en.htm


[23]http://europa.eu/legislation_summaries/environm 

ent/nature_and_biodiversity/l28046_en.htm 

106


BIODIVERSITY CONSERVATION IN CONSTANŢA COUNTY 

Silvia TURCU*, Marcela POPOVICI**, Loreley JIANU** 

*Ovidius University of Constanţa, Doctoral School, Biology Domain, 

Mamaia Avenue, No. 124, Constanţa, 900552, Romania, sscturcu@yahoo.com 

** Environmental Protection Agency Constanţa, Unirii Street, No. 23, Constanţa, 900532 

__________________________________________________________________________________________ 

Abstract: Nature conservation is the action taken by human society to maintain and perpetuation of species of 

plants and animals. Recognition of the value of biodiversity in Constanta County is done by the special 

protection of habitats and species for an important number of protected areas. The main instrument governing the 

activities taking place at the perimeter and adjacent of natural areas is management plan of protected area, in 

accordance with existing environmental legislation. 

Keywords: biodiversity conservation, protected areas, administration and custody, Constanţa County. 

__________________________________________________________________________________________ 


Nature Conservation is the action taken by 

human society to maintain and perpetuation of 

species of plants and animals [1]. In our country, to 

provide special measures of protection and 

biodiversity conservation, was instituted a tiered 

system of protection, conservation and use, 

according to the following categories of protected 

areas: national interest (scientific reserves, national 

parks, natural monuments, nature reserves, natural 

parks), the international interest (natural sites of 

universal natural heritage, geoparks, wetlands of 

international importance, biosphere reserves), the 

community interest or Natura 2000 sites (sites of 

Community Importance, Special Areas of 

Conservation Areas Special Protection Bird) or 

local interest [2]. 


In Constanţa County, there are over 900 

species spermatophytes present, most of these are 

characteristic species of steppe and forest steppe 

habitats, over 200 species of vascular flora of 

national interest, with varying degrees of 

vulnerability, some of these are endemic species 

[3]. 

Fauna of Constanţa County is characterized 

by great wealth, represented by more than 345 

vertebrate taxa - 45 species of mammals, 243 birds, 

19 species of reptiles, 10 species of amphibians and 

28 species of fish - and a significant number of 

invertebrates [3]. 

In Constanţa County, natural and semi-natural 

habitats, found in all environments (aquatic, 

terrestrial and subterranean), are classified into 

seven classes (coastal and halophilic communities, 

continental water, scrub and grassland, forests, 

marshes and wetlands, screes, rock and continental 

sands and agricultural land and artificial 

landscapes) which include 58 types of natural 

habitat and ruderal communities (agricultural land 

and artificial landscapes) [4]. 

Thus, since 1970, a number of valuable areas 

in terms of biodiversity were declared reserves by 

decisions of Constanţa County People’s Council. 

In 2000, only two of the previously declared 

protected natural areas remains areas of local 

interest (Table 1), for the rest of these, by Law 

5/2000 [5] is nationally recognized protected area 

status. 

In coming years, new laws were imposed on 

other areas of protected area status of national 

interest: Government Decision 2151/2004 [6], 

Government Decision 1581/2005 [7], Government 

Decision 1143/2007 [8] and currently totaling 36 

protected natural areas of national interest (Table 

2). 

Since joining the European Union in 2007, 

Romania has emerged as one of the nations that 

have a true natural heritage, with many protected 


Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010) 

areas and many species listed in Annexes of Birds 

and Habitats Directives. Under European 

Directives, European Council Directive 92/43 EEC 

[9], and Birds Directive - European Council 

Directive 79/409 EEC [10], countries of European 

Union (EU) ensures maintenance or restoration of 

natural habitats and wild fauna and flora of 

Community interest in a favorable conservation 

status, to help maintain biodiversity. 

Following the transposition of these two 

Directives into national law was established system 

of protection for 42 areas: 22 special protection 

areas for birds (SPA), reported by Government 

Decision no. 1284/2007 [11] and a number of 20 

sites of community importance (SCI), declared by 

Order no. 1964/2007 [12] (Table 3 and Table 4). 

There is a part of the “Danube Delta” Biosphere 

Reserve, internationally protected area, on 

administrative territory of Constanţa County. This 

is the largest protected area in the country and has a 

threefold international status: Biosphere Reserve, 

Ramsar Site and Site of World Natural and Cultural 

Heritage (Table 4). “Danube Delta” Biosphere 

Reserve has its own administrative structure 

established by Law 82/1993 [13]. Management plan 

af this protected area was developed by Danube 

Delta “Biosphere Reserve” Administration. 

Techirghiol Lake became the Ramsar Site on 

March 23, 2006 and was classified as wetland of 

international importance by Government Decision 

no. 1586/2006 [14] (Table 4). In addition to this 

status, Techirghiol Lake was declared nature 

reserve and Bird Protection (Natura 2000). This 

protected area has not been attributed in custody, 

but “Dobrogea-Litoral” Water Directorate, in 

partnership with The Romanian Ornithological 

Society have developed a management plan for 

Lake Techirghiol trough project 

LIFE04NAT/RO/000220 Improving wintering 

condition for Branta ruficollis at Techirghiol Lake. 

As can be seen from Tables 1, 2, 3, 4 and 5 

responsibilities for managing natural protected 

areas, placed under special protection and 

conservation, belong to local authorities for 

protected natural areas declared by decisions of 

their, to “Danube Delta” Biosphere Reserve 

Administration for Biosphere Reserve Danube 

Delta and to custodians/ administrators for natural 

protected areas declared by law, by Government 

108 

decisions or by order of the central public authority 

for environmental protection. Gaining of 

custody/administration of natural protected areas is 

in accordance with the procedure of Government 

Decision 1533/2007 [15]. However, within six 

months of the signing of custody agreement for 

natural protected areas, custodian must develope 

regulation of protected area, which contains the 

rules will be respected within the protected area, 

and within a year to effectuate the protected area 

management plan, in line with regulation. The 

measures provided in management plans of 

protected natural areas are developed taking 

account of economic requirements, social and 

cultural as well as on regional and local area, but 

with priority for the objectives which led to the 

establishment of protected area. 


Recognition of the value of biodiversity in 

Constanta county is done by the special care and 

protection of habitats and species for a number of 

two protected areas of county interest, 36 protected 

natural areas of national interest, 42 protected 

natural areas of interest (Natura 2000 sites): 22 of 

Special Protection Areas for Birds (SPAs) and 20 

Sites of Community Importance (SCI), two natural 

areas of international concern. 

Currently, of the 82 protected areas in the 

county of Constanţa 68 are administered according 

to law, and 14 will be assumed to custody until the 

end of 2010. 

Conservation of biodiversity is in accordance 

with existing environmental legislation and 

management plan of protected areas is the main 

instrument governing the activities taking place at 

the perimeter and adjacent natural areas. 

Management of protected natural areas in 

Constanţa County will improve by developing the 

management plans, by custodians/ administrators. 

4. References 

[1] BAVARU A. et al., 2007- Biodiversitatea şi 

ocrotirea naturii, Editura Academiei Române. 

[2] ***Government Emergency Ordinance no. 

57/2007 on the regime of natural protected areas, 

natural habitats, flora and fauna.

Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010) 

[3] ***Report on the Environmental Conditions in 

Constanţa County in 2009. 

[4] DONIŢĂ N. et al. 2005, "Habitatele din 

Romania", Editura Tehnică şi Silvică. 

[5] ***Law 5/2000 approving the national spatial 

plan and is nationally recognized and protected 

area status. 

[6] ***Government Decision 2151/2004 on the 

establishment of protected area regime to new 

areas, 

[7] Government Decision 1581/2005 concerning 

the establishment of protected area system to new 

areas. 

[8] ***Government Decision 1143/2007 

concerning the establishment of new protected 

areas. 

[9] ***European Council Directive 92/43 EEC on 

the conservation of natural habitats and wild flora 

and fauna adopted on May 21, 1992. 

[10] ***European Council Directive 79/409 EEC 

on the conservation of wild birds taken on April 

2, 1979. 

109 

[11] ***Government Decision no. 1284/2007 

declaring Bird specially protected areas as part of 

the European ecological network Natura 2000 in 

Romania. 

[12] ***Order of Ministry of Environment and 

Sustainable Development no. 1964/2007 

concerning the establishment of protected area 

system of sites of Community importance, as part 

of European ecological network Natura 2000 in 

Romania. 

[13] ***LAW no 82/1993 establishing Biosphere 

Reserve “Danube Delta”. 

[14] ***Government Decision no. 1586/2006 on 

the classification of protected areas in the 

category of wetlands of international importance. 

[15] ***Order no. 1533/2008 approving the 

Methodology for the award of administration of 

natural protected areas that require the 

establishment of administrative structures and 

methodology for awarding custody of protected 

natural areas that do not require the creation of 

management structures.


Table 1. Natural Protected Areas of county interest 

No. Protected area Administrator 

1. “Arborele Corylus colurna” - natural monument Constanţa Hall 

2. “Pâlcul de stejari brumării” - natural monument Mangalia Hall 

Table 2. Natural Protected Areas of national interest 

No. Protected area Administrator/ Custodian 

1. 

“Acvatoriul litoral-marin Vama Veche-2 Mai” 

- Zoological and Botanical Reserve 

- 

2. 

“Canaralele din Portul Hârşova” - 

Morfogeological Monument 

National Forest Administration ROMSILVA- 

Forestry Department Constanţa 

3. 

“Cetatea Histria” Scientific Reserve - 

Archaeological Site part of Danube Delta 

“Biosphere Reserve” 

Danube Delta “Biosphere Reserve” 

Administration -Tulcea 

4. 

“Dealul Alah Bair” - Complex Nature 

Reserve 



5. 

“Dunele marine de la Agigea” - Botanical 

Nature Reserve 

A.I. Cuza University - Iaşi 

6. 

“Grindul Chituc” - Scientific Reserve part of 



Administration - Tulcea 

7. 

“Grindul Lupilor” - Scientific Reserve part of 



Administration - Tulcea 

8. “Gura Dobrogei” - Complex Nature Reserve 



9. “Lacul Agigea” - Zoological Nature Reserve - 

10. “Lacul Bugeac” - Complex Nature Reserve 



11. “Lacul Dunăreni” - Complex Nature Reserve 



12. “Lacul Oltina” - Complex Nature Reserve 



“Lacul Techirghiol” - Zoological Nature 

13. 

Reserve 

- 

14. “Lacul Vederoasa” - Complex Nature Reserve 



“Locul fosilifer Aliman” - Paleontological 

15. 

Monument 



“Locul fosilifer Cernavodă”- Geological and 

16. 

Paleontological Monument 



“Locul fosilifer Credinţa” - Paleontological 

17. 

Monument 



“Locul fosilifer Movila Banului”- Geological 

18. 

and Paleontological Monument 



110


“Recifii jurasici Cheia” - Geological and 

19. 

Botanical Nature Reserve 

“Mlaştina Hergheliei” - Complex Nature 

20. 

Reserve 

“Obanul Mare şi Peştera ” - 

21. Speleological and Morfogeological Nature 

Reserve 

22. “Pădurea Bratca” - Complex Nature Reserve 

“Pădurea Canaraua-Fetii”- Botanical and 

23. 

Zoological Nature Reserve 

“Pădurea Celea Mare - Valea lui Ene” - 

24. 

Complex Nature Reserve 

25. “Pădurea Cetate” - Complex Nature Reserve 

“Pădurea Dumbrăveni” - Botanical and 

26. 


“Pădurea Esechioi” - Botanical and Zoological 

27. 


“Pădurea Fântâniţa-Murfatlar” - Botanical and 

28. 


“Pădurea Hagieni” - Botanical and Zoological 

29. 


30. 

“Pereţii calcaroşi de la Petroşani” - Geological 

Monument 

“Peştera ” - Speleological 

31. 

Monument 

“Peştera ” - Scientific 

32. 

Speleological Reserve 

“Peştera ” - Speleological 

33. 

Monument 

“Reciful neojurasic de la Topalu” - Geological 

34. 

and Paleontological Monument 

“Corbu-Nuntaşi-Histria” – Scientific Reserve 

35. 

part of Danube Delta “Biosphere Reserve” 

“Valu lui Traian Rezervaţie” - Archaeological 

36. 

and Botanical Nature Reserve 

Table 3. Special Protection Areas – for Birds (SPA) 

111 



The Group of Underwater and Speleological 

Exploration - Bucharest 































No. Site Name Administrator/ Custodian 

1. “Aliman – Adamclisi” 



2. “Allah Bair – Capidava” 



-


3. 

“Balta Mică a Brăilei” 


Forestry Department Brăila 

4. 

“Balta Vederoasa” 



5. 

“Băneasa - Canaraua Fetei” 



6. 

“Canaralele de la Hârşova” 



7. 

“Cheile Dobrogei” 



8. “Delta Dunării şi Complexul Razim – Danube Delta “Biosphere Reserve” 

Sinoie” 


9. 

“Dumbrăveni” 



10. 

“Dunăre – Ostroave” 



11. “Dunărea Veche - Braţul Măcin” - 

12. 

“Lacul Bugeac” 



13. 

“Lacul Dunăreni” 



14. 

“Lacul Oltina” 



15. “Lacul Siutghiol” - 

16. “Lacurile Taşaul – Corbu” - 

17. “Lacul Techirghiol” - 

18. 

“Limanu – Herghelia” 



19. “Marea Neagră” EUROLEVEL 

20. 

“Pădurea Hagieni” 



21. “Stepa Casimcea” - 

22. “Stepa Saraiu – Horea” - 

Table 4. Sites of Community Importance (SCI) 

No. Site Name Administrator/ Custodian 

1. “Balta Mică a Brăilei” National Forest Administration ROMSILVA- 

Forestry Department Brăila 

2. “Braţul Măcin” - 

3. “Canaralele Dunării” National Forest Administration ROMSILVA- 


4. “Dealul Alah Bair” National Forest Administration ROMSILVA- 


5. “Delta Dunării” Danube Delta “Biosphere Reserve” 

Administration 

6. “Delta Dunării - zona marină” Danube Delta “Biosphere Reserve” 

Administration 

112


7. “Dumbrăveni - Valea Urluia - Lacul National Forest Administration ROMSILVA- 

Vederoasa” 


8. “Dunele marine de la Agigea” A.I. Cuza University Iasi 

9. “Fântâniţa Murfatlar” National Forest Administration ROMSILVA- 


10. “Izvoarele sulfuroase submarine de la 

Mangalia” 

GEOECOMAR 

11. “Mlaştina Hergheliei - Obanul Mare şi The Group of Underwater and Speleological 

Peştera Movilei” 


12. “Pădurea Esechioi - Lacul Bugeac” National Forest Administration ROMSILVA- 


13. “Pădurea Hagieni - Cotul Văii” National Forest Administration ROMSILVA- 


14. “Pădurea şi Valea Canaraua Fetii – National Forest Administration ROMSILVA- 

Iortmac” 


15. “Peştera Limanu” The Group of Underwater and Speleological 


16. “Plaja submersă Eforie Nord - Eforie Sud” EUROLEVEL 

17. “Podişul Nord Dobrogean” - 

18. “Recifii Jurasici Cheia” National Forest Administration ROMSILVA- 


19. “Vama Veche - 2 Mai” - 

20. “Zona marină de la Capul Tuzla” 

GEOECOMAR 

Table 5. Natural Protected Areas of international concern 

No. Protected Area Administrator 

1. “Lacul Techirghiol” - Ramsar Site - 

2. 

“Delta Dunării” – Biosphere Reserve, Ramsar Site, 

World Heritage Site Natural and Cultural 

Danube Delta “Biosphere 

Reserve” Administration -Tulcea 

113


THE PRESENT SITUATION OF THE NOSE HORNED VIPER POPULATIONS 

(VIPERA AMMODYTES MONTANDONI BOULENGER 1904) 

FROM DOBRUDJA (ROMANIA AND BULGARIA) 

Marian TUDOR 

Universitatea Ovidius Constanţa, Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole 

B-dul Mamaia, nr. 124, Constanţa, 900527, România, e-mail 

__________________________________________________________________________________________ 

Abstract: Due to the destruction and deterioration of the specific habitats and the increased fragmentation of the 

remaining ones, the nose horned viper has lost large tracts of vital living space. In addition, road kills, direct kills 

and collecting by humans contribute to their decline. I tried to estimate the present situation of the nose horned 

viper populations in Dobrudja, based on literature and our own field data. The main goals were: to investigate the 

present situation of the nose horned populations in Dobrudja; to identify the most suitable habitats for Vipera 

ammodytes montandoni; and to locate the viable populations of this viper and current threats to the nose horned 

viper populations. 

Keywords: Dobrudja, Nose-Horned Viper, viable populations 

__________________________________________________________________________________________ 


The study of the nose horned viper in general, 

and of the Dobrudja subspecies in particular, can 

offer both herpetologists and conservationist 

biologists important data due to the relatively strict 

habitat requirements of this herpeto-taxon (particular 

habitat conditions, the necessary presence of certain 

prey-species in the habitat etc), as well as to its 

vulnerability to the modifications of the specific 

habitats. From this point of view, it is one of the ideal 

species for monitoring in the protected areas, as well 

as in those territories to be designated protected areas 

in the future. The subspecies is considered critically 

endangered (CR) in the Vertebrates Red List of 

Romania [1] and it is included in annex 3A of OM 

1198/2005 (Species of European interest in need of 

strict protection, critically endangered species). 

The populations of Vipera ammodytes 

montandoni are in a continuous decline [2] due to 

anthropogenic causes and their need for preservation 

is all the more imperative as the destruction of the 

specific habitats has increased considerably over the 

last few years. 


Starting with 1995, thirty-eight locations 

mentioned in literature ([3], [4], [5], [6], [7], [8], [9]) 

in the Romanian area of Dobrudja have been 

explored with the purpose of verifying the 

preservation state of the nose horned viper 

populations. Eight more locations have been explored 

for the same reason in the Bulgarian region of 

Dobrudja in 2008. 

The researches took place especially in spring 

and autumn, when the vipers are more active and 

more easily recognizable in the specific habitats [10], 

[11], [12], [13], [14]. The explorations used visual 

transects as well as the method of active search in the 

specific habitats. [11], [15]. 

The capture and handling of the vipers was 

accomplished with the help of the herpetological 

hook and tongs ([16], [17]). Leather gloves were used 

in the case of small individuals. After identification 

and sex determination, each individual was released 

in the same place where it was captured from. Also, 

the roads that bordered or intersected the explored 

habitats were repeatedly examined, and all the road 

kills were photographed and collected. The searches 

led many times to the discovery of individuals whose 

death was a result of the direct interaction with 


The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010) 

humans. In such cases, the vipers had usually been hit 

to death with stones or other 

hard objects. No instances of natural death were 

identified among the dead individuals. 

All the inventoried individuals in each 

researched habitat were quantified and the 

determination of the viability degree of the 

populations was attempted by means of calculating 

the identified adult/juveniles proportion [18]. The 

calculation of the viability degree also took into 

account the state of the habitats and particularly the 

level of human intervention, starting from the premise 

that a natural or semi-natural habitat offers much 

better conditions for the survival of a nose horned 

viper population than an anthropogenic one. 


The study has rendered evident certain aspects 

that complete the data regarding the state of the nose 

horned viper populations in Dobrudja. Thus, if before 

our researches, it was considered that Vipera 

ammodytes montandoni has a relatively large 

distribution in Dobrudja [7], [19], [9], our data rather 

bring arguments in favor of the idea that this 

subspecies currently occupies small habitats in more 

or less strictly delineated areas. This aspect supports 

the idea that the exchanges of individuals among 

populations are very poor or lack completely. This 

may lead in time to the reduction of the intrapopulation 

genetic diversity. 

Also, most of the habitats of nose horned viper 

populations in Dobrudja are intersected or bordered 

by roads. Thus, it was observed that, out of a total of 

thirty-eight locations situated in the Romanian part of 

Dobrudja where populations of nose horned viper had 

previously been mentioned [7], only in twenty-five of 

them (65.8%) the presence of this herpeto-taxon 

could be rendered evident. Numerous monasteries 

and hermitages have been built over the past ten years 

and their presence already generates a rise in the 

number of direct kills in some locations where there 

were populations of nose horned viper such as 

Babadag Forest, Gura Dobrogei, Dumbraveni, 

Hagieni and the foot of Pricopanului Peak. 

The existence of this Dobrudja subspecies could 

no longer be evidenced in the other thirteen locations 

116 

previously mentioned as habitats for nose horned 

viper populations. 

The study has evidenced the fact that in 

Dobrudja (both the Romanian and the Bulgarian 

side), the largest populations of Vipera ammodytes 

montandoni are situated in Dumbraveni Natural 

Reserve, Babadag Forest, Priopcea Hill, Macin 

Mountains National Park, Gura Dobrogei Natural 

Reserve, the ruins of Adamclisi fortress, Canaraua 

Fetii Natural Reserve, Rusalka, Kaliakra, Bolata 

Dèrè, Yaillata and Kamen Bryag. Of the total 

inventoried individuals in the researched areas, 14% 

were represented by animals whose death was a result 

of the anthropogenic impact. Among these, 67% are 

represented by vipers killed deliberately, most having 

a crushed skull, and 33% are road kills, especially in 

spring when these reptiles prefer to bask directly on 

road asphalt (figure 1). 

67% 

33% 

Fig. 1. The raport Road kill/Direct kill 

road kills 

direct kills 

The estimation of population viability in 

Dumbraveni Natural Reserve, Babadag Forest, 

Priopcea Hill, Macin Mountains National Park, Gura 

Dobrogei Natural Reserve, the ruins of Adamclisi 

fortress, Canaraua Fetii Natural Reserve, Rusalka, 

Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag 

evidenced the fact that the number of juveniles 

compared to that of adults is relatively high in these 

areas, which could thus indicate a high viability of 

these populations. As a whole, the situation is 

graphically illustrated in figure 2. 

In what regards the abundance of individuals in 

the researched populations, it was observed that in 

locations such as Gura Dobrogei, Babadag, 

Dumbraveni, Adamclisi, Hagieni, Canaraua Fetii and 

Bolata Dèrè, the number of identified individuals is 

higher (figure 3). Still, this aspect only leads to the

Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010) 

conclusion that these populations might be larger than 

the ones identified and investigated. Also, this aspect 

must be correlated with the number of field hours 

spent in each location. If the number of field 

hours spent in the Romanian Dobrudja is 

approximately equal (generally over 100 hours) in 

each location, the number of hours spent in the 

locations of the Bulgarian Dobrudja is much lower 

(an average of 10-12 hours per location). This is why 

it is very likely that the number of individuals in the 

identified populations could be much higher in the 

Bulgarian locations. Considering the time spent in 

each location, the relative preservation of the 

habitats, as well the effort of capturing the animals, 

all these bring arguments in favor of this hypothesis. 

Otherwise, in what regards these populations in 

the Bulgarian side of Dobrudja, the data collected 

over the 2008 research season evidence a relatively 

good preservation of the nose horned viper in the 

natural and semi-natural habitats. No road kills or 

direct kills were discovered in these areas, probably 

due to the fact that these habitats are located at a 

considerable distance from roads and spaces 

dedicated to activities with anthropogenic impact. 

Still, given that the data collected here were gathered 

over a period of only a few months, it is possible that 

direct kills could occur sporadically due to tourism or 

animal grazing [20]. 

At the same time, we estimate that the new 

buildings, as well as the sale of lands that shelter 

vipers to investors, will lead to the destruction of 

their specific habitats in Bulgaria too. In both 

countries, the expansion of constructions and road 

improvement with the purpose of easing transport but 

also of facilitating the access of mass tourism to wild 

areas, will lead to the enhancement of the 

anthropogenic impact in areas where it either did not 

exist or it was sporadic. 


The main conclusion of the study is that 

Dobrudja, as biogeographical area well 

circumscribed and with particular characteristics 

compared to the other parts of Europe situated at the 

same latitude, still hosts viable populations of the 

montandoni horned viper subspecies; 

117 

The most serious danger for the preservation of 

this subspecies of horned viper is represented by the 

destruction of habitats. Immediately after come the 

road kills and direct kills. 

The populations of Vipera ammodytes 

montandoni in Dobrudja are isolated one from the 

other, therefore we believe that there are few 

exchanges of individuals among them or that these 

exchanges lack completely in some cases, leading 

thus to the reduction of the intra-population genetic 

diversity; 

Our data argument for the existence of at least 

12 areas that shelter viable populations of nose 

horned vipers in Dobrodja. These areas are: 

Dumbraveni Natural Reserve, Babadag Forest, 

Priopcea Hill, Macin Mountains National Park, Gura 

Dobrogei Natural Reserve, the ruins of Adamclisi 

fortress, Canaraua Fetii Natural Reserve, Rusalka, 

Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag. 

Future studies will focus on the estimation of 

intra-population genetic diversity and on the 

dynamics of certain populations of this subspecies in 

order to propose the best measures intended for the 

preservation of the Dobrudja nose horned viper. 

Acknowledgements 

This study was partly possible thanks to the 

UNDP/GEF Atlas Project no. 047111 “The 

strengthening of the national system of protected 

areas in Romania through the best management 

practices in the Macin Mountains National Park.” 

The researches in the Bulgarian area of 

Dobrudja were possible thanks to the PHARE CBC 

2005 Romania-Bulgaria Program RO 2005/017- 

535.01.02.02 “Comparative studies regarding the 

biodiversity of coastal habitats, the anthropogenic 

impact and the possibilities for the conservation of 

important European habitats between Cape Midia 

(Romania) and Cape Kaliakra (Bulgaria). 

We are indebted to: 

Dr. Dan Cogălniceanu and Dr. Marius Skolka 

for providing references, valuable advice and 

logistics. 

Dr. Zsolt Török and Dr. Paul Szekely for 

support and references. 

Dr. Olivia Chirobocea for the revising of the 

text and accurate English translation.


5. References 

[1] IFTIME, A. (2005) - Reptile. In: Cartea Roșie a 

vertebratelor României, 173–196. 

BOTNARIUC ,N. & TATOLE, V. (Eds.). 

Bucuresti: ed. Curtea Veche.[in Romanian]. 

[2] GIBBONS, J.W. et. al. 2000 - The Global 

Decline of Reptiles, Déjà Vu Amphibians 

BioScience, Vol 50, No.8, 653-666. 

[3] TOROK, Z.1999 - Note privind distribuția 

spațiala a herpetofaunei în zona Culmii 

Pricopanului, Acta oecologica, 6, 57-62. 

[4] TOROK, Z. 2000 - Șerpii veninoși din România 

(Venomous snake of Romania), Petarda, no.6, 

Tulcea, Aves. 

[5] SOS, T., 2005 - Note preliminare privind 

distribuția spațiala a herpetofaunei de pe 

Culmea Pricopanului din Parcul Național Munții 

Măcin, Migrans, Târgu Mureș, 7(3), 8-10. 

[6] OTEL, V., 1997 - Investigatii herpetologice în 

zona munților Măcin și podișul Babadagului, 

Anal Șt. IDD, 1997, 71-77. 

[7] FUHN, I.E. & VANCEA, St. 1961 - Reptilia 

(Țestoase, Șopirle, Șerpi). In: Fauna RPR.Vol. 

14(2). Bucuresti: Edit. Academiei RPR. 338 [in 

Romanian]. 

[8] MERTENS, R. & WERMUTH, H. (1960) - Die 

Amphibien und Reptilien Europas. Dritte 

Liste,nach dem Stand vom 1. Januar 1960. 

Frankfurt am Main: Verlag Waldemar Kramer. 

264. 

[9] COVACIU-MARCOV S.D, GHIRA I., 

CICORT-LUCACIU A.D., SAS I., 

STRUGARIU A., BOGDAN H.V. - 

Contributions to knowledge regarding the 

geographical distribution of the herpetofauna of 

Dobrudja, Romania. North-Western Journal of 

Zoology Vol. 2, No. 2, 2006, 88-125 

[10] FUHN, I.E. (1969) - Broaste, serpi, sopirle. 

Bucuresti: Ed. Natura si Omul. 246 [In 

Romanian]. 

[11] COSSWHITE, D.L., S.F. FOX, and THILL 

R.E. 1999 - Comparison of methods for 

monitoring reptiles and amphibians in upland 

forests of the Ouachita mountains, Proceedings 

of the Oklahoma Academy of Science 79:45-50. 

118 

[12] CAMPBELL, H.W., and S.P. CHRISTMAN 

1982 - Field techniques for herpetofaunal 

community analysis. 193-200 in N. J. Scott, Jr., 

ed. Herpetological Communities, U.S.D.I. Fish 

and Wildlife Service, Wildlife Research Report 

13, Washington, D.C. 239 . 

[13] RYAN, T.J., PHILIPPI, T., LEIDEN, Y.A., 

DORCAS, M.E., WIGLEY, T.B. and 

[14] GASC, J.-P., CABELA, A., CRNOBRHJA- 

ISAILOVIC, J.,DOLMEN, D., 

GROSSENBACHER, K., HAFFNER, 

P.,LESCURE, J., MARTENS, H., MARTINEZ- 

RICA, J.P., MAURIN, H., OLIVEIRA, M.E., 

SOFIANIDOU, T.S., VEITH, M. & 

ZUIDERWIJK, A.(Eds.) 1997 - Atlas of 

Amphibians and Reptiles in Europe. 

[15] ENGE, K.M. 2001 - The pitfalls of pitfall traps. 

Journal of Herpetology 35(3): 467-478. 

[16] FERNER, J. W. 1979. A review of marking 

techniques for amphibians and reptiles, Society 

for the Study of Amphibians and Reptiles, 

Circular 9: 1-42. 

[17] KARNS, D.R. 1986 - Field herpetology: 

methods for the study of amphibians and 

reptiles, in Minnesota. James Ford Bell 

Museum of Natural History, occasional papers 

18. 

[18] CORN, P. S., and R. B. BURY. 1990 - 

Reptiles. USDA Forest Service, General and 

Technical Report PNW-GTR-256, 34. 

[19] ANDREI, M., 2002 - Contributions to the 

knowledge of the herpetofauna of southern 

Dobrudja (Romania). Trav. Mus. Nat. d'Hist. 

Nat. Gr. Antipa 44, 357-373 

[20] BOIAN, P.P. 2007 © Springer, Amphibians and 

Reptiles of Bulgaria: Fauna, Vertical 

Distribution and Consrvation, 85-107 , in 

Biogeography and Ecology of Bulgaria, V. Fet 

& A. Popov (eds.)


Rusalka 

Yailata 

Kamen Bryag 

Kaliacra 

Bolata Dèrè 

Canaraua Fetii 

Hagieni 

Adamclisi 

Dumbrăveni 

Babadag 

Gura Dobrogei 

Măcin 

Priopcea 

Niculiţel 

Târguşor 

Cerna 

Albesti 

Şipotele 

Casimcea 

Atmagea 

Beştepe 

Cataloi 

0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 

119 

Juveniles 

Adults 

Fig. 2. The percentage of adults and juveniles in the analyzed populations

Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010) 

Canaraua Fetii 

Hagieni 

Adamclisi 

Dumbrăveni 

Babadag 

Gura Dobrogei 

Măcin 

Yailata 

Bolata Déré 

Kaliakra 

Priopcea 

Rusalka 

Kamen Bryag 

Niculiţel 

Târguşor 

Cerna 

Albesti 

Şipotele 

Casimcea 

Atmagea 

Beştepe 

Cataloi 

0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 16.00% 

Fig. 3. The abundance of Nose-Horned Viper in the analyzed populations 

120


BODY SIZE VARIATION IN RANA TEMPORARIA POPULATIONS 

INHABITING EXTREME ENVIRONMENTS 

Rodica PLĂIAŞU ** , Raluca BĂNCILĂ ** , Dan COGĂLNICEANU * 

* Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences, Aleea Universităţii nr. 1, 

corp B, Constanţa 900470, Romania 

** “Emil Racoviţă” Institute of Speleology, 13 Septembrie Road, No. 13, Bucharest 050711, Romania 

___________________________________________________________________________ 

Abstract: We studied the variation in body size in populations of a widespread anuran species, Rana 

temporaria, from high altitude and latitudes. Our results indicated a variable interannual pattern of body size, 

suggesting that body size in extreme environments is influenced by many factors. This indicates that long-term 

series of observations are needed to separate natural fluctuations from man-induced changes. 

Keywords: Rana temporaria, extreme environments, body size, interannual variation 

__________________________________________________________________________________________ 


During the last decades, many amphibian 

species have declined from high altitude area, even in 

habitats apparently without human impact [1, 2]. The 

causes of some declines remain unknown. 

Understanding of the life history characteristics of 

the amphibian populations that inhabit extreme 

environments at high altitude and latitude is an 

important step in the evaluation process of the 

potential causes of decline. Genetic and 

environmental factors (e.g. temperature, rainfall, 

trophic resources, competition, predators) determine 

variation in the life history traits of species 

occupying a large geographic area [3]. Low 

temperature, associated with high altitude/latitude, 

reduces the activity period and the time available for 

resource exploitation [4]. Temperature affects the 

duration of hatching and metamorphosis in 

amphibians. The increase in the adult body size has 

been frequently associated with a cold annual 

temperature [5, 6]. Most studies of variation in 

amphibians body size have focused on latitudinal and 

altitudinal variation, e.g. trying to establish if the 

amphibian species follow the Bergmann’s rule [7, 8]. 

Studies on the interannual variations in amphibians 

body size generally analyze difference in body 

condition [9, 10], or variation in age and size at 

maturity [6]. 

The Common Frog (Rana temporaria) is the 

most widespread amphibian species in Europe [11]. 

Its distribution reaches 71 o N in Fennoscandia [12] 

and it can be found even at altitudes of 2600 m [12]. 

The wide altitudinal and latitudinal range of this 

species, allows comparisons of life-history traits over 

a broad range of conditions. In a previous publication 

we analyzed the altitudinal and latitudinal body size 

variation among populations from high altitude and 

latitude of R. temporaria testing if the variation 

pattern is according to the Bergmann’s rule [13]. In 

this study we analyzed interannual body size 

variation in the same Rana temporaria populations, 

in order to evaluate if the pattern of variation in body 

size changes in time. We tested the following 

predictions: i) there is no interannual variation in 

body size and ii) the mean body size of the frog 

populations from subarctic regions shows significant 

variation during a growth season. 


R. temporaria populations were studied from 

Kilpisjarvi, Finland (latitude N 69 o ) in 2003 (August) 

and 2009 (July), Kolari, Finland (latitude 67.2 o ) in 

2009 (July) and in Retezat National Park, Romania 

(latitude N 45 o ) in 2004 (September) and 2009 

(August). Latitude and altitude were recorded for 

each population by using a handheld Garmin GPS. 


Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) 

Captured individuals were sexed, weighed (W) 

to the nearest 0.01 g with a portable electronic 

balance (AccuLab Pocket Pro), and snout-vent length 

(SVL) was measured to the nearest 0.5 mm with dialcalipers. 

Data were log transformed prior to analyses. 

For comparisons between years and sites we used 

One-way analysis of variance (ANOVA) and 

Analysis of covariance (ANCOVA) to compare the 

slopes of the regression lines. 

Statistical analyses were performed using SPSS ver. 

10.0 (SPSS Inc., 1999). 


A total of 347 individuals were measured and 

weighed in 2003/2004, of which 237 juveniles and 

110 adults (67 males and 43 females) and 157 

individuals in 2009 (66 juveniles, 43 females and 48 

males). Both log transformed W and SVL were 

normally distributed (W: D = 5.06, p < 0.001; 

122 

SVL: D = 3.75, p < 0.001). The body size indices of 

the studied populations are presented in Tables 1 and 

2. There was no significant difference in SVL 

between Retezat National Park and Finland - 

Kilisjarvi populations. We found significant 

differences in the mean body size indices between 

the two stations from Finland (Table 3). 

We then compared W and SVL from different years 

for the same population. We found significant 

differences in the interannual variation of the body 

size indices for juveniles in both Finland and Retezat 

populations, and in the mean weight for females. 

Males showed only in Retezat a significant 

interannual variation in the body size indices (Table 

4). We also compared the slopes of the regression 

lines of W as a function of SVL. The slopes of the 

regression lines are significantly different for all 

adults in Retezat and Finland (Fig. 1: F1,56= 81.41, 

p

Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) 

Table 3. Comparison of SVL and W between the three R. temporaria populations, by using ANCOVA (FN = 

Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; N = sample size, *P < 0.05, ***P < 

0.001, NS = not significant). 

W SVL 

N Average Fa Average Fa 

FN vs FS FN FS FS FN FS FN 

Female 13 16 18.956 14.308 6.069* 71.244 61.154 26.384*** 

Male 17 9 17.267 15.853 0.737 NS 

69.89 62.46 22.88*** 

Juveniles 50 9 4.333 7.779 10.381* 42.17 45.95 1.748 NS 

FN vs RNP FN RNP FN RNP FN RNP 

Female 13 14 14.308 34.69 19.504*** 61.154 76.78 3.627 NS 

Male 17 22 15.853 36.20 17.93*** 62.46 73.41 1.778 NS 

Juveniles 50 7 7.779 9.28 5.903* 45.95 44.8 0.198 NS 

Table 4. Comparison of the interannual variation in SVL and W between the three R. temporaria populations, by 

using ANCOVA (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitude; N = sample 

size, *P < 0.05, ***P < 0.001, NS = not significant). 

W SVL 

N Average Fa Average Fa 

FN 2003 vs. 2009 2003 2009 2003 2009 2003 2009 

Female 29 13 31 14.308 4.235* 65.94 61.154 3.70 NS 

Male 32 17 31.76 15.853 3.713 NS 

67.59 62.46 0.43 NS 

Juveniles 197 50 3.17 7.779 78.41*** 29.01 45.95 120.01*** 

RNP 2004 vs. 2009 2004 2009 2004 2009 2004 2009 

Female 14 14 61.16 34.69 14.56*** 82.7 76.78 2.24 NS 

Male 35 22 47.14 36.20 12.21*** 77.07 73.41 4.49* 

Juveniles 40 7 2.19 9.28 22.56*** 24.42 44.8 19.4*** 

The variation in the adult body size reported in 

amphibians can be induced by several factors, 

including genetic and environmental differences, 

such as: duration of the activity period, food 

availability and climatic conditions [6, 14]. Laugen 

et al. (2005) found that body size decreased with 

latitude in the Scandinavian Common Frog 

populations. Comparisons between populations from 

Western Europe with different activity periods report 

increases in the mean length, as activity period gets 

shorter [6]. Rana temporaria populations from the 

analyzed area show a variable pattern in weight and 

length. Băncilă et al. (2010) found that latitudinal and 

altitudinal variation patterns in juvenile body size 

123 

were according to the Bergmann’s rule. We found an 

opposite pattern for juveniles, with decreases in the 

body size as the activity period gets shorter. Since 

juveniles have higher growth and development rate 

than adults, difference could be observed even in the 

case of short periods of time between sampling. 

Interpopulational variation in the adult body size 

could be caused by differences in the age structure. 

Growth rates in amphibian species can dramatically 

decrease after the attainment of sexual maturity (e.g. 

Miaud et al. 1999). Thus, delayed reproduction can 

allow a prolonged growth period and the attainment 

of a larger adult size.


W (log) 

2.0 

1.8 

1.6 

1.4 

1.2 

1.0 

1.75 1.80 1.85 1.90 1.95 2.00 

SVL (log) 

RNP 2004 

RNP 2009 

Fig. 1. Body size indices for males in RNP 

populations, 2004 (N=35; R 2 = 0.72) and 2009 (N=22; 

R 2 = 0.90). 

W (log) 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 

1.65 1.70 1.75 1.80 1.85 1.90 1.95 

SVL (log) 

Kilpisjarvi 2003 


Fig. 3. Body size indices for females in Finland- 

Kilpisjarvi, 2003 (N=29; R 2 = 0.63) and 2009 (N=13; 

R 2 = 0.59). 

W (log) 

1.6 

1.5 

1.4 

1.3 

1.2 

1.1 

1.0 

0.9 

Kolari 

Kilpisjarvi 

1.70 1.75 1.80 1.85 1.90 1.95 

SVL (log) 

Fig. 5. Body size indices for females in Finland 

Kilspijarvi (N=13; R 2 = 0.59) and Finland Kolari 2009 

(N=16; R 2 = 0.71). 

124 

W (log) 

2.2 

2.0 

1.8 

1.6 

1.4 

1.2 

1.0 

1.70 1.75 1.80 1.85 1.90 1.95 2.00 2.05 

SVL (log) 

RNP 2004 

RNP 2009 

Fig. 2. Body size indices for females in RNP 

populations, 2004 (N=14; R 2 = 0.75) and 2009 (N=14; 

R 2 = 0.95). 

W (log) 

1.8 

1.6 

1.4 

1.2 

1.0 

0.8 



1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 

SVL (log) 

Fig. 4. Body size indices for males in Finland- 

Kilpisjarvi, 2003 (N=32; R 2 = 0.70) and 2009 (N=17; 

R 2 = 0.63). 

W (log) 

1.5 

1.4 

1.3 

1.2 

1.1 

1.0 

0.9 

Kolari 

Kilpisjarvi 

1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 

SVL (log) 

Fig. 6. Body size indices for males in Finland 

Kilpisjarvi (N=17; R 2 = 0.89) and Finland Kolari 2009 

(N=9; R 2 = 0.70).

Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010) 

Factors such as temperature and humidity can 

directly affect the activity period and the 

availability of food, influencing the growth rate and 

the fat stores; hence they could consequently 

determine significant interannual variation in the 

body size. Populations from both analyzed areas 

exhibit interannual variation in weight and length. 

This variation mainly affects the weight and could 

be the result of the differences in the sampling 

period. The pattern of the adult size variation could 

also directly result from the variation in the 

population age structure. Further analyses are 

necessary to determine whether variation in the age 

structure are contributing or not to the interannual 

body size indices. Results suggest that many factors 

affect the body size in extreme environment and 

long-term series of observations are needed in order 

to separate natural fluctuations from the human 

impact/global warming. 


This study stresses the importance of analyzing 

interannual variation of life history traits, because 

one-year data may not properly reflect the features 

of a population and this issue becomes important in 

the context of global changes and their possible 

effects on the amphibian populations. 

Acknowledgements 

The research was funded by the EU FP6 

(Lapbiat) and EU FP7 (Lapbiat 2) Romanian 

CNCSIS grant 1114/2004. We are grateful to 

Claudia Jianu, Dorel Ruşti, Ioan Ghira and Marian 

Tudor for their help during fieldwork. 

5. References 

[1] LAURANCE W.F., McDonald K.R., Speare R., 

1996 - Epidemic disease and the catastrophic 

decline of Australian rain forest frogs. 

Conservation Biology, 10: 406-413. 

[2] YOUNG B.E., Lips K.R., Reaser J.K., Ibanez 

R., Salas A.W., Cedeno J.R., Coloma L.A., Ron 

S., La Marca E., Meyer J.R., Munoz A., Bolanos 

F., Chaves G., Romo D. 2001. Population 

declines and priorities for amphibian 

125 

conservation in Latin America. Conservation 

Biology, 15: 1213-1223. 

[3] SORCI G., Clobert J., Belichon S., 1996 - 

Phenotypic plasticity of growth and survival in 

the common lizard Lacerta vivipara. Journal of 

Animal Ecology, 65: 781-790. 

[4] RYSER J., 1996 - Comparative life histories of 

a low- and a high-elevation population of the 

common frog Rana temporaria. Amphibia– 

Reptilia, 17: 183-195. 

[5] FICETOLA G.F., Scali S., Denoël M., 

Montinaro G., Vukov T.J., Zuffi M.A.L., Padoa- 

Schioppa E., 2010 - Ecogeographical variation of 

body size in the newt Triturus carnifex: 

comparing the hypotheses using an informationtheoretic 

approach. Global Ecology and 

Biogeography, 19: 485-495. 

[6] MIAUD C., Guyétant R., Elmberg J., 1999 - 

Variations in life-history traits in the common 

frog (Rana temporaria) (Amphibia: Anura): a 

literature review and new data from the French 

Alps. Journal of Zoology, 249: 61-73. 

[7] ADAMS D.C., Church J.O., 2008 - Amphibians 

do not follow Bergmann’s rule. Evolution, 62: 

413-420. 

[8] ASHTON K.G., 2002 - Do amphibians follow 

Bergmann’s rule? Canadian Journal of Zoology, 

80: 708-716. 

[9] TOMAŠEVIĆ N., Cvetković D., Aleksić I., 

Crnobrnja-Isailović J., 2007 - The effect of 

climatic conditions on post-hibernation body 

condition and reproductive traits of Bufo bufo 

females. Archives of Biological Sciences, 

Belgrade, 59: 51-52. 

[10] TOMAŠEVIĆ N., Cvetković D., Miaud C., 

Aleksić I., Crnobrnja-Isailović J., 2008 - 

Interannual variation in life history traits between 

neighbouring populations of the widespread 

amphibian Bufo bufo. Revue d’Ecologie (Terre et 

Vie), 63: 371-381. 

[11] GASC J.P., Cabela A., Crnobrnja-Isailovic J., 

Dolmen D., Grossenbacher K., Haffner P., 

Lescure J., Martens H., Martínez Rica J.P., 

Maurin H., Oliveira M.E., Sofianidou T.S., Veith 

M., Zuiderwijk A. (ed), 1997 - Atlas of 

Amphibians and Reptiles in Europe. Collection 

Patrimoines Naturels, 29, Societas Europaea 

Herpetologica, Muséum National d'Histoire


Naturelle & Service du Petrimone Naturel, Paris, 

496 pp. 

[12] MERILÄ J., Laurila A., Laugen A.T., 

Rasanen K., Pahkla M., 2000 - Plasticity in age 

and size at metamorphosis in Rana temporaria - 

comparison of high and low latitude populations. 

Ecography, 23: 457-465. 

[13] BĂNCILĂ R.I., Plăiaşu R., Cogălniceanu, D., 

2010 - Effect of latitude and altitude on body size 

in the common frog (Rana temporaria) 

126 

populations. Studii şi Cercetări, Biologie, 

Universitatea din Bacău,17: 43-46. 

[14] MORRISON C., Hero J., 2002 - Geographic 

variation in life history characteristics of 

amphibians: a review. Journal of Animal 

Ecology, 72: 270-279. 

[15] LAUGEN A.T., Laurila A., Jönsson K.I., 

Söderman F., Merilä J., 2005 - Do common frogs 

(Rana temporaria) follow Bergmann’s rule? 

Evolutionary Ecology Research, 7: 717-731.


UTILIZATION OF EPIFLUORESCENCE MICROSCOPY AND DIGITAL IMAGE 

ANALYSIS TO STUDY SOME MORPHOLOGICAL AND FUNCTIONAL ASPECTS 

OF PROKARYOTES 

Simona GHIŢĂ ** , Iris SARCHIZIAN * , Ioan ARDELEAN *** 

* Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology, 

Mamaia Avenue, No. 124, Constanţa, 900552, Romania, 

e-mail: ghitasimona@aim.com, irissarchizian@yahoo.com 

** Constanta Maritime University, Department of Environmental Engineering, Mircea cel Batrin, No. 104, 

Constanta, 900663, Romania, e-mail:ghitasimona@aim.com; 

*** Institute of Biology, Splaiul Independenţei, No. 296, Bucharest, 060031, Romania, 

email:ioan.ardelean57@yahoo.com 

__________________________________________________________________________________________ 

Abstract: The aims of this study is to argue, based on original results, the importance of utilization of 

epifluorescence microscopy to study some morphological and functional aspects of prokaryotes allowing to 

perform total cell counts , direct viable count count, count of permeabilised cells, chlorophyll containing cells or 

putatively capsulated cells. Automated image analysis of the results thus obtained was done using CellC and 

ImageJ software which allow the quantification of bacterial cells from digital microscope images, automated 

enumeration of bacterial cells, comparison of total count and specific count images, providing also quantitative 

estimates of cell morphology. 

Keywords: epifluorescence, digital image analysis, heterotrophic bacteria, cyanobacteria. 

__________________________________________________________________________________________ 


The use of epifluorescence microscopy to study 

different aspects of prokaryotes at population and 

single cell level significantly improved the 

knowledge concerning which species are present in a 

given sample, the cell density and the metabolic 

statues of the population as a whole or of each single 

prokaryote cell (Van Wambeke, 1995; Manini & 

Danovaro, 2006; Falcioni et al., 2008; Kirchman, 

2008; Ardelean et al., 2009). In the last decades there 

is also an increase in the development and use of 

different softwares for automated analysis of the 

digital images thus obtained (Ishii et al. 1987; Estep 

& Macintyre 1989; Embleton et al., 2003; Walsby, 

1996; Congestri et al. 2003; Selinummi et al., 2005, 

2008). 

The aims of this study is to argue, based on 

original results, the importance of utilization of 

epifluorescence microscopy coupled with automated 

image analysis to study some morphological and 

functional aspects of prokaryotes allowing to 

perform total cell counts (acridine orange, DAPI, 

SYBR Green 1), direct viable count (elongated cell in 

the presence of nalidixic acid, labelled with acridine 

orange), count of permeabilised cells (cells 

permeable to propidium iodide), putatively 

capsulated cell (labelled with aniline blue) and 

chlorophyll containing cells both in enriched cultures 

and in natural (microcosms) samples. 


A. Study area and sampling. Samples were 

collected in sterile bottles in October 2008 and May 

2009 from sulphurous mesothermal spring (Obanul 

Mare) placed in north of Mangalia City 

(43˚49’53.6’’N; 28˚34’05.3’’E). The samples were 

divided in sub-samples, one being immediately fixed 

with buffered formaldehyde (2% final concentration) 

and the second one used to isolate cyanobacteria by 

inoculation into conical flasks with either BG11 


Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) 

medium or nitrate - free BG11 medium (BG0) 

(Rippka et al., 1979). Another series of natural 

samples were collected from Black Sea (Tomis 

seaport at 0.5m depth; 44 o 10 ’ 44 ’’ N; 28 o 39 ’ 32 ’’ E) in 

March 2009. 

B. Culture conditions. Natural samples 

inoculated in either BG11 or BG0 media, either solid 

of liquid, were incubated in culture room at 25 ± 1ºC 

and illuminated with fluorescent tubes having the 

photon rate of 50 μmol m –2 s –1 at surface of the 

culture vessels. 

C. Microcosms. Taking into account the 

advantages of microcosms (Iturbe et al., 2003; 

Molina-Barahona et al., 2004) we used this 

opportunity as previously (Ardelean et al., 2009). 

D. Total cell count (AO; DAPI, SYBR Green 

I) 

Total bacterial count were performed using 

acridine orange, DAPI and SYBR Green I (Luna et 

al., 2002; Lunau et al., 2005; Manini & Danovaro, 

2006). 

For AO and DAPI (5 μg/mL dye final 

concentrations) subsamples were stained for 5 

minutes and were filtred on black Millipore 0,22µm 

pore size filters. Unlike AO, using DAPI for bacterial 

visualization and enumeration has the advantages of 

low background fluorescence and that DAPI stains 

only DNA. 

For SG (1µL/10µL sample final concentrations) 

subsamples were stained for 10 minutes and were 

filtred on black Millipore 0,22 µm pore size filters. 

Color filters were washed with 10 ml of 17 ‰ saline 

solution. SG as a permeant DNA-binding stain and 

determine the total fraction of cells from natural 

samples. 

E. Permeabilized (dead) cells (PI+) 

PI is a double-charged phenanthridium 

derivative and is one of the most common stains for 

dead cells (Luna et al., 2002). PI is thus assumed to 

be unable to penetrate cell membranes. In our natural 

samples we used a PI concentration of 5 μL/ml 

sample. Also stained samples were filtered through 

black Millipore 0,22 µm pore size filters and then 

inspected under a epifluorescence microscope. The 

disruption of planktonic cell aggregates for cell 

enumeration were done as previously shown 

(Ardelean et al., 2009). 

128 

F. Enumeration of (putatively) capsulated 

cells (AB+). Cell capsule was also inspected using 

aniline blue (AB) which is a fluorescent dye specific 

which seems to be specific for 1,3 beta glucans 

(Hong et al., 2001) found in plants and as capsular 

material in many microorganisms (Nakanishi et al., 

1976; McIntosh et al., 2005). Capsular envelopes are 

widely distributed in marine free-living and particleassociated 

bacteria (Heissenberger et al., 1996) and 

are a signature of active bacteria (Stoderegger & 

Herndl, 2002). Bacteria with an intact intracellular 

structure, and therefore potentially active bacteria, 

are surrounded by a capsular layer, while the vast 

majority of bacteria with a damaged structure lack 

such a capsule (Heissenberger et al., 1996). 

Laboratory experiments indicated that active bacteria 

are constantly renewing their capsular envelope and 

releasing a significant fraction of the polysaccharide 

layer into the ambient water (Stoderegger & Herndl, 

2002). The samples were treated with AB (5 µg/mL 

final concentration) for 5 minutes and then filtered 

and counted as shown above for AO staining . 

G. The automatic cell analysis were done with 

two software ImageJ and CellC, who was applied to 

digital images of whole cells color-stained bacteria 

and cyanobacteria. The analysis proceeds few 

important steps: the background is separated from the 

objects based on the intra-class variance threshold 

method; noise and specks of staining color in the 

image can affect the reliability of the analysis, so 

those was removed. The removal was done applying 

mathematical morphology operations to the image; 

then separation of clustered objects was performed 

(Selinummi, 2008). The length of cells was 

determined with ImageJ software using a calibration 

scale. 

H. Cyanobacteria (natural fluorescence) 

Visualization of hydrocarbon tolerant 

phototrophic microorganisms, also for unicellular or 

filamentous cyanobacteria from sulphurous 

mesothermal spring; chlorophyll a in natural 

environments (either marine or spring) was done 

using an epifluorescence microscope (N-400FL, lamp 

Hg 100W, type on the blue filter; Sherr et al., 2001) 

as previously shown (Ardelean et al., 2009). 

I. Direct viable count (cells capable of 

division) is based on the Kogure method developped

Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010) 

by incubation of samples with a single antimicrobial 

agent (nalidixic acid) and nutrients (yeast extract). 

Nalidixic acid acts as a specific inhibitor of DNA 

synthesis and prevents cell division without affecting 

other cellular metabolic activities, including cell 

growth; thus viable cells growth but do not divide, 

thus becoming longer/larger than cells unable to grow 

(Kogure et al., 1979). Experiments were done with 

40mL samples from each microcosms in which the 

sample was filtered through 0.45 µm filter (2 and 3) 

supplemented with yeast extract (50 mg / L final 

concentration), nalidixic acid (20 mg / L final 

concentration) (Kogure et al., 1979) and gasoline 

(0.5% final concentration); 17 hours before the start 

of experiment all samples were kept in an incubator 

at a temperature of 30 o C and continuous stirring. 

Subsequently samples were incubated under the 

conditions previously reported and samples were 

harvested each two hours (considering the time To, 

T1 –after 2 hours, T2- after 4 hours; T3- after 6 hours, 

T4- after 8 hours). 


1. Total count cell (AO+, DAPI+), permeable 

(dead) cells (PI+) and (putative) capsulated cells 

(AB+) 

In experimental microcosms we viewed the 

gasoline tolerant/oxidizing bacteria to make a clear 

distinction between the total number of cells (stained 

with AO, DAPI), the number of encapsulated, active 

cells, (AB +), and the number of permeable (PI+) , 

dead (figures 1 and 3). 

Fig 1. Comparison between the total cell count (AO+ 

and DAPI+) and permeable cells density (IP+) 

129 

As shown in figure 1 the total number of 

heterotrophic cells counted using AO or DAPI is 

practically the same. Quantification was performed 

on samples previously fixed in experimental 

microcosms (M1 and M2). Comparing the total 

number of bacterial cells obtained with AO and 

DAPI stained (20 μL/mL sample) in experimental 

microcosms, we have shown that there are no 

significant differences in the use of two 

fluorochromes on natural samples (M1: 13.719,5 

cells ml -1 – SD (±39,7) AO and 13.494,6 cells ml -1 - 

SD (±22,2) DAPI, respectively M2: 14.619,1 cells 

ml -1 – SD (±28,8) AO and 15.787,4 cells ml -1 SD 

(±32,8) DAPI). 

Fig 2. Total number of cells obtained using AO and 

SG in natural sample 

To assess the number of cells obtained after 

staining AO and SG, we used unfixed samples 

collected from microcosm 1. 

As shown in figure 2 there are differences in 

total counts obtained by the use of either AO or 

SYBR Green 1, the higher count obtained with the 

last fluorochrome (47,6%) being due to its higher 

fluorescence yield, in agreement with international 

literature (Weinbauer et al., 1998; Luna et al., 2002; 

Lunau et al., 2005 ), allowing the visualization of 

smaller cells. 

As shown in figure 1, the number of dead cells 

(PI positive) is 12.3% of the total number obtained 

with the two fluorochromes, AO and DAPI. 

In figure 3 one can see the cell densities of 

putative capsulated cells which are 10% from the 

density obtained with acridine orange.


Fig 3. Aniline blue positive cells as compared with 

acridine orange positive cells. 

2. Cyanobacteria (natural fluorescence) 

Natural fluorescence of these prokaryotic in 

various natural environments (marine and sulphurous 

mesothermal spring) and marine microcosms was 

studied by epifluorescence microscopy (figure 4). 

a b 

c d 

Fig 4. Natural fluorescence of gasoline-tolerant 

oxygenic phototrophic microorganism from 

microcosm 2 supplemented with gasoline (a); 

microcosm 1 supplemented with gasoline and 

nutrient (b) and microorganism isolated from 

sulphurous mesothermal spring Obanul Mare 

(Mangalia) (c and d). 

In microcosm 1 cyanobacteria filaments are 

much thinner (1.35 ± 0.27) compared with 

microcosm 2 (3.87 ± 0.57) (fig.5); the significance of 

this difference being under investigation. 

130 

Fig 5. Filaments of cyanobacteria in the M2. 

3. Direct viable count 

In figure 6 there are presented the results 

concerning changes in average cell lengths of 

bacterial populations from the two microcosms with 

filtered water (0,45µm) each supplemented with yest 

extract, nalidixic acid and gasoline (see Materials and 

methods). 

Fig 6. Average length of cells from To to T4 (after 8 

hours of incubation with nalidixic acid) in the two 

microcosms (M2 and control, M3) 

As can be seen in Figure 6, after 8 hours of 

incubations, the average length of M2 cells is about 7 

µm, compared with the M3 where the cells were 

maintained in high proportion in the form of cocci 

(average diameter of about 2 µm). These results 

argue the possibility to count viable cells, cells able 

to grow, by a relatively simple method. It seems 

appropriate to assume that the large increase in cell 

size in bacterial populations which have been 

previously selected to grow in the presence of 

gasoline (microcosms 2) is due to the cells ability to 

oxidize/tolerate gasoline, as compared with the 

populations sampled from the control microcosms 

where the proportion of gasoline tolerant bacteria is


very low (and responsible for the low increase in the 

average cell lengths in M3). In M3 (control) one can 

see a rather constant length of some cells during 

incubation (2,15±0,37) whereas in M2 there was a 

sudden increase in cell length (6,46±1,54) to the time 

T2 (4 hours incubation) then there was a steady 

increase until T4 (8 hours incubation). 

In Figure 7 are some random fields of cells in 

the two microcosms to highlight how cell elongation 

occurred from the T o to T4 only in M2. 

a b 

c d 

Fig 7. Evaluating cell elongation in microcosm 2 (a- 

To and b-T4) respectively in the control microcosm 

(c- To and d-T4) 

Digital Image Analysis and automated image 

analysis for epifluorescence 

The automated approach will not only remove 

the need for tedious manual analysis work, but also 

enable biologists to measure cellular features not 

feasible by the standard manual techniques 

(Selinummi, 2008). 

In our studies we used ImageJ software - a 

public domain Java image processing and analysis 

program inspired by NIH Image for the Macintosh, 

who runs, either as an online applet or as a 

downloadable application, on any computer with a 

Java 1.5 or later virtual machine. This software was 

used to display, edit, analyze, process, save and print 

8–bit, 16–bit and 32–bit epifluorescence digital 

images, many image formats including TIFF, GIF, 

JPEG, BMP, supporting ‘stacks’and hyperstacks, a 

series of images that share a single window. 

131 

For study bacteria and cyanobacteria from our 

samples ImageJ was the main software for measure 

the length of cells and pixel value statistics of userdefined 

selections, creating density histograms and 

line profile plots, supports standard image processing 

functions such as contrast manipulation, sharpening, 

smoothing, edge detection and median filtering. 

Digital images are two-dimensional grids of 

pixel intensities values with the width and height of 

the image being defined by the number of pixels in x 

(rows) and y (columns) direction. Thus, pixels 

(picture elements) are the smallest single components 

of images, holding numeric values – pixel intensities 

– that range between black and white (ImageJ user 

guide). Microphotographs used in this study was 

RGB images, RGB/HSB stacks, and composite 

images. 

People can see color with significant variations 

and the popular phrase “One picture is worth ten 

thousand words” may not apply to certain color 

images, especially those that do not follow the basic 

principles of Color Universal Design. That why this 

combining digital image analysis and automated 

analysis methods was usefull to distinguish some 

morphological and functional aspects of prokaryotes. 

We displied with ImageJ simultaneously several 

selections or regions of interest named ROIs, who can 

be measured, drawn or filled. Selections was initially 

outlined in one of the nine ImageJ default colors 

(Red, Green, Blue, Magenta, Cyan, Yellow, Orange, 

Black and White) and then, once created, selections 

was contoured or painted with any other color. Most 

of ImageJ analyses was printed to the Results table. 

Fig 8. The ImageJ Window 

(http://rsbweb.nih.gov/ij/). 

Straight Line Selection with “Alt” from 

computer keeps the line length fixed while moving 

either end of the line and forces the two points that 

define the line to have integer coordinate values when 

creating a line on a zoomed image. 

The CellC software is the second software used 

in automated analysis of our microscopy images like 

cell enumeration and measurements of cell’s 

properties (size, shape, intensity). We applied the


algorithms of CellC software for digital images, 

because this have three important parts: a MATLAB 

figure file of the segmented image (this can be 

exported in any common image file format; a comma 

separated value (CSV) - file with quantitative data of 

the cells (was opened in a spreadsheet program Excel 

for further analysis); a summary CSV-file with the 

cell count for each of the analyzed images for a quick 

overview of the analysis process (this file were only 

saved in the batch processing mode). Fluorescence 

microscopy digital images were analyzed and the 

objects has different intensity than the background. 

Commonly, this property holds true for images of 

bacteria (http://sites.google.com/site/cellcsoftware/). 

Fig 9. CellC’s interface 

(http://sites.google.com/site/cellcsoftware/) used for 

automated digital analysis of bacteria/cyanobacteria. 

Furthermore, CellC software were used for two 

important purposes: to calculate total object count 

(e.g. DAPI stained cells) and co-localization analysis, 

comparing total and specific count images of the 

same location. When two images were analyzed, the 

co-localization was measured by comparing which 

cells are present only in the first image, and which are 

visible in both of the images. The binarized result 

images was saved as JPG-images, and the 

enumeration results and statistics are saved as an 

Excel-ready CSV file. The images was processed 

one at a time, or automatically in a batch. 

Graphical illustration of the analysis process 

and a part of a CSV-file opened in a spreadsheet 

program are given in Figure 9. The CSV-file gives, 

for each cell in the image, size and intensity 

information as well as information on cell 

morphologies. All results produced with digital image 

processing algorithms are perfectly reproducible. 

The image processing methods used guarantee 

that all images are analyzed using the same criteria, 

and therefore results between different images are 

comparable. CellC software is easy to use due to the 

132 

included graphical user interface, and the batch 

processing mode enables fast and convenient 

processing of hundreds of cell images. 

CellC enumerate bright cells on a dark 

background (epifluorescence). We also used two 

different methods to process the images: one 

image/image pair at a time; several images pairs 

sequentially in batch processing mode. 

If the background of the image is uneven (because of 

e.g. misaligned lighting), it is preferable to choose 

this option. 

The default option in CellC is to present the 

measured parameters in pixels. By checking this box 

we define how many micrometers one pixel 

corresponds to, and receive all measurement results 

in micrometers. The correct value of this setting 

obviously depends on the imaging setup, such as on 

the camera and the objective, and must be determined 

outside CellC, using ImageJ to calibrate the scale. 

The main technical requirement for using CellC 

is the clear visual distinction between the cells to be 

counted and their background, which could be 

achieved relatively easy by epifluorescence 

microscopy (Ardelean et al., 2009). 

If darker regions exist inside cells, thresholding 

may result in false holes inside cells (darker pixels 

are considered background). By selecting this option, 

these holes are automatically filled. Sometimes the 

fill can cause worse cell cluster separation results. 

Automatic removal of over/undersized cells 

were selected, because CellC automatically decides 

which particles are too small to be considered as real 

cells. All detected objects that are smaller than 1/10 

of the mean size of all objects, were removed. 

Because the sizes of under/oversized particles were 

known using “Analyze Measure” option of ImageJ, it 

was possible to set the thresholds manually by using 

the text boxes. The unit of sizes depends on the user 

defined unit (pixels/μm 2 ). 

The CSV data sheet consists of following 

columns: cell's serial number (a unique number given 

to each cell); area of cell (estimate of the cell area); 

approximate volume (approximation of the volume of 

the cell); length (estimate of the cell length); width 

(estimate of the cell width); intensity mean, (mean 

intensity of the cell); intensity maximum, (maximum 

intensity of the cell); solidity (estimate of the shape of 

the cell); compactness (estimate of the shape of the


cell). Means of each column and the unit of measure 

(pixels or micrometers) are presented in the end of 

the file. 

Image acquisition—Images for analysis were 

done with a Canon digital camera. Brightness and 

contrast were adjusted for the first image and kept 

unchanged throughout the image acquisition 

procedure. The images (1600 by 1200 pixels, 256 

dpi) were acquired at 50x magnification and stored as 

543-KB JPG files. Additional images acquired at 

100x magnification were used to verify that 

measurements of individual filaments/ bacteria were 

independent of magnification 

a) Acridin-orange stained filamentous 

cyanobacteria isolated from mesothermal sulfurous 

spring were analysed using Image J software for 

distinguish heterocystous cells. First of all, the 

original RGB image (Figure 10 A) were transformed 

into 32-bit images, then we adjust the 

brightness/contrast and also applied smooth or find 

edges (Figure 10 B) option from processing images. 

The same image were analysed with CellC software 

(Fig. 10 C) to count the cells from filamentous 

cyanobacteria or to measure the size of each cells. 

A B 

C 

Fig 10. A – digital image of heterocystous 

cyanobacteria isolated from sulphurous mesothermal 

spring Obanul Mare (Mangalia) stained with AO; B – 

find edges of panel A using ImageJ software; C- total 

count analysis of panel A using CellC software (48 

cells counted from cyanobacteria’s filaments). 

Validation of any count were done using 

manual count. 

ImageJ software were used for 

automated measuring cell’s length (µm), using a 

133 

calibrated eyepiece graticule as reference (Ardelean 

et al, 2009). 

Digital images from AO staining filaments of 

cyanobacteria in microcosm were treated with ImageJ 

to distinguish the heterocystous cell. 

Fig 11. Cyanobacteria with heterocyst presence in 

microcosm 2; AO staining (arrow indicate heterocyst 

cell present in samples of microcosm supplemented 

with gasoline). 

To avoid uncertain estimates of filament length 

and width, the number of filaments presented in one 

image should not be too high. Extreme filament 

densities would undoubtedly increase filament 

overlap and lead to uncertain measurements unless 

samples are diluted (Almesjö & Rolff, 2007). 

We use a blue light epifluorescence filter set to 

visualize AO-stained bacteria (N-400FL type). AO 

stains both DNA and RNA so is used for the 

enumeration of total bacteria. 

In figure 12 A we present only an example of 

digital analysis of fig.7 b: first, we adjust 

contrast/brightness of digital image, then analyse 

measure of graticula presented in fig. 7b and set the 

calibration bar to determine correctly the length of 

each bacteria treated with nalidixic acid. In B is 

presented image analysis using CellC software. 

A B 

Fig 12. Image analysis program Image J (A) and 

CellC (B) from microcosm 2, elongated cells at time 

T4 (8hours) 

b) DAPI were used to view filamentous 

cyanobacteria isolated from mesothermal sulfurous


spring and heterotrophic and phototrophic bacteria 

from marine environment. 

The fluorochrome DAPI is the most commonly 

bacterial stain for a wide range of sample types. 

DAPI is a nonintercalating, DNA-specific stain which 

fluoresces blue or bluish-white (at or above 390 nm) 

when bound to DNA and excited with light at a 

wavelength of 365 nm (Kepner & Pratt, 1994). When 

unbound, or bound to non-DNA material, it may 

fluoresce over a range of yellow colors (see 

figure13). DAPI-stained filaments of cyanobacteria 

isolated from sulphurous spring Obanul Mare 

(Mangalia) reveal heterogeneous cells as can be seen 

in Fig.13a. 

a 

b 

Fig 13. DAPI-stained cyanobacteria isolated from 

sulphurous spring (a), arrows indicates septa between 

cells ; bacteria/cyanobacteria isolated from marine 

environment (b); both a and b treated with Image J 

and CellC software. 

Fig 14 . Cyanobacteria and heterotrophic cells in 

microcosm supplemented with gasoline/M2 - DAPI 

stain 

134 

c) Aniline blue is highly specific for staining 

type polysaccharide. Use of aniline blue is a good 

method not only for detection of production of 

exocellular β-1,3-glucan, but also for detection of 

some β-glucan in the cell wall (Nakanishi et al., 

1976). In figure 15 is apparent the AB stained 

heterotrophic cells from microcosm supplemented 

with gasoline and cyanobacteria cells from 

microcosms and sulphurous spring samples. 

a b c 

Fig 15 . Visualisation of encapsulated bacteria and 

cyanobacteria after aniline-blue staining on M2 (a), 

M1 (b) and from sulphurous spring samples (c). 

d) PI staining is generally used for the 

evaluation of plasma membrane integrity by 

fluorescence. Literature mentions that molecular 

weighs PI is 668,4 and is thus assumed to be unable 

to penetrate cell membrane (Manini & Danovaro, 

2006). In figure16 living bacteria appeared green due 

to the excitation of the AO dye with which the cells 

have been stained and the samples stained with PI 

and appear red fluorescent cells; the bacteria were 

counted under blue excitation. 

a b 

c 

Fig 16. Marine bacteria examined using 

epifluorescence microscopy (magnification x1000), 

Fig (a) illustrate bacteria stained with propidium 

iodide (dead cells) in microcosms 1 and (b) 

respectively microcosms 2; total count analysis using 

the CellC software (c).


e) Natural fluorescence - In figure 17 we 

describe succesfully separatation with ImageJ of cells 

by natural fluorescence of photosynthetic gasolinetolerant/oxidant 

microorganisms isolated from 

mesothermal sulphurous spring in different chanell – 

red and green- and then each image were automat 

counted with CellC, obtaining finally the number of 

cells red and green separately. 

Fig 17. Natural fluorescence of (A) photosynthetic 

gasoline-tolerant/oxidant microorganisms isolated 

from mesothermal sulphurous spring and digital 

image analysis of chlorophyll autofluorescence in (B) 

green channel ; (C) red channel and (D-E) total count 

analysis of red/green channels using the CellC 

software. 

In figure 18 there are presented images showing 

the natural fluorescence of chlorophyll, as an image 

of marine oxygenic gasoline tolerant/ oxidant 

phototrophic microorganisms. Difference is clearly 

apparent width of filaments of cyanobacteria 

developed in the experimental microcosms. The 

measurements were performed with Image J program 

as shown previous (see point 2). 

a b 

Fig 18. Autofluorescence of chlorophyll from 

oxygenic photosynthetic microorganisms: 

microcosms 1 (a) and 2 (b). 

Epifluorescence techniques and image analysis 

has increasingly been used to determine cell size, 

bacterial abundance and detection of physiological 

characteristics like damaged versus intact cell 

membranes. 

135 

Throughout the investigations conducted 

continuously attempted to determine the nature of 

connections between communities of microorganisms 

and how and to which condition each. 


The utilization of epifluorescence microscopy 

and digital image analysis enable us to study some 

morphological and functional aspects of prokaryotes: 

total cell counts (acridine orange, DAPI, SYBR green 

1), direct viable count (elongated cell in the presence 

of nalidixic acid, labelled with acridine orange), 

count of permeabilised cells (cells permeable to 

propidium iodide), capsulated cell (labelled with 

aniline blue) and chlorophyll containing cells, both 

in enriched cultures and in natural / microcosms 

samples. 

The total number of heterotrophic cells counted 

using AO or DAPI is practically the same whereas 

total counts obtained with SYBR Green 1 are 47,6% 

higher. 

The number of dead cells (PI positive) and that 

of (putative) capsulated cells are 12.3% and 10%, 

respectively of the total number ( AO and DAPI). 

The image analysis systems presented here was 

performed for counting and estimating the length of 

bacteria/cyanobacteria with uniform morphology. 

The presented methods does not totally exclude 

the need for manual microscope analyses of water 

samples, and automated procedures must 

intermittently be validated by independent manual 

procedures. 

Acknowledgment 

We are grateful to Dr. Tech. Jyrki Selinummi 

(Department of Signal Processing, Tampere 

University of Technology, Finland) for very useful 

and kind advices concerning the use of software 

CellC and ImageJ. 

5. References 

[1] VAN WAMBEKE F., 1995- Numeration et taille 

des bacteries planctoniques au moyen de 

lànalise dìmages couplee a lèpifluorescence. 

Oceanis, 21: 113-124.


[2] MANINI E. & DANOVARO R., 2006- Synoptic 

determination of living/dead and active/dormant 

bacterial fraction in marine sediments. FEMS 

Microbiol. Ecol, 55: 416-423. 

[3] FALCIONI T., PAPA S., GASOL J.M., 2008- 

Evaluating the flow-cytometric nucleic acid 

double-staining protocol in realistic situations of 

planktonic bacterial death. Appl. Environ. 

Microbiol, 74: 1767-1779. 

[4] KIRCHMAN D.L., 2008- Microbial Ecology of 

the Oceans. John Wiley & Sons, NY, 593. 

[5] ARDELEAN I.I., GHIŢĂ S., SARCHIZIAN I., 

2009-Epifluorescent method for quantification 

of planktonic marine prokaryotes, Proceedings 

of the 2 nd International Symposium “New 

Research in Biotechnology” serie F: 288-296. 

[6] ISHII T., ADACHI R., OMORI M., SHIMIZU 

U., IRIE H., 1987 - The identification, counting, 

and measurement of phytoplankton by an 

image-processing system. J. Cons. Ciem., 

43:253-260. 

[7] ESTEP K. W. & MACINTYRE F., 1989 - 

Counting, sizing, and identification of algae 

using image analysis. Sarsia, 74: 261-268. 

[8] EMBLETON K. V., GIBSON C. E., HEANEY S. 

I., 2003 - Automated counting of phytoplankton 

by pattern recognition: a comparison with a 

manual counting method. J. Plankt. Res., 25: 

669-681. 

[9] WALSBY A. E. & AVERY A., 1996 - 

Measurement of filamentous cyanobacteria by 

image analysis. J. Microbiol. Meth., 26:11-20. 

[10] CONGESTRI R., CAPUCCI E., ALBERTANO 

P., 2003 – Morphometric variability of the 

genus Nodularia (Cyanobacteria) in Baltic 

natural communities. Aquat. Microb. Ecol. 32: 

251-259. 

[11] SELINUMMI J., SEPPÄLÄ J., YLI-HARJA O., 

PUHAKKA J.A., 2005 - Software for 

quantification of labeled bacteria from digital 

microscope images by automated image 

analysis”. BioTechniques, 39: 859–863. 

[12] SELINUMMI J., 2008- On Algorithms for Two 

and Three Dimensional High Throughput Light 

Microscopy, Ph.D Thesis for the degree of 

Doctor of Technology. 

[13] RIPPKA R., DERUELLES J., WATERBURY 

J.B,. HERDMAN M., STANIER R.Y., 1979 - 

136 

Generic assignments, strain histories and 

properties of pure cultures of cyanobacteria. 

Journal of Geneneral Microbiology., 111: 1 - 

61. 

[14] ITURBE R., FLORES R.M., TORRES L.G., 

2003- Subsoil polluted by hydrocarbons in an 

out-of-service oil distribution and storage 

station in Zacatecas, Mexico. Environ Geol, 44: 

608–620. 

[15] MOLINA-BARAHONA L., RODRI´GUEZ- 

VA´ZQUEZ R., HERNAŃDEZ- VELA´SCO, 

C. VEGA-JARQUIN, O. ZAPATA-PE´REZ 

M., MENDOZA- CANTU´ A., ALBORES A., 

2004- Diesel removal from contaminated soils 

by biostimulation and supplementation with 

crop residues, Appl Soil Ecol, 27: 165–175. 

[16] LUNA G.M., MANINI E., DANOVARO R., 

2002- High fraction of dead and inactive 

bacteria in coastal marine sediments: 

comparison of protocols and ecological 

significance. Appl. Environ. Microbiol, 68: 

3509-3513. 

[17] LUNAU M., LEMKE A., WALTHER K., 

MARTENS-HABBENA W., SIMON M., 2005- 

An improved method for counting bacteria from 

sediments and turbid environments by 

epifluorescence microscopy. Appl. Environ. 

Microbiol, 7: 961-968. 

[18] HONG Z., DELAUNEY A.J., VERMA D.P.S., 

2001-A cell plate-specific callose 

synthase and its interaction with 

phragmoplastin.Plant Cell, 13:755-768 

[19] NAKANISHI I., KAZUTSUGU K., SUZUKI 

T., ISHIKAWA M., BANNO I., SAKANE T., 

HARADA T.,1976- Demonstration of curdlantype 

polysaccharide and some other β-1, 3- 

glucan in microorganisms with aniline blue. 

J. Gen. Appl. Microbiol., 22: 1-11. 

[20] MC INTOSH M., STONE B. A.,. STANISICH 

V. A,, 2005-Curdlan and other bacterial (1-3)-β- 

D-glucans. Appl Microbiol Biotechnol, 68: 163- 

173. 

[21] HEISSENBERGER A., LEPPARD G.G., 

HERNDL G.J., 1996 - Relationship between the 

intracellular integrity and the morphology of the 

capsular envelope in attached and free-living 

marine bacteria. Appl. Environ. Microbiol., 62: 

4521-4528.


[22] STODEREGGER K.E. & HERNDL G.J., 2002 - 

Distribution of capsulated bacterioplankton in 

the North Atlantic and North Sea. Microb. 

Ecol., 44: 154-163. 

[23] SHERR B., SHERR E.B., DEL GIORGIO P., 

2001- Enumeration of total and highly active 

bacteria. Methods in Microbiology, 30: 129- 

161. 

[24] KOGURE K., SIMIDU U., TAGA N., 1979- A 

tentative direct microscopic method for 

counting living marine bacteria. 

Can.J.Microbiol, 25:415-420. 

[25] WEINBAUER M.G., BECKMANN C., HOFLE 

M.G., 1998 – Utility of green fluorescent 

nucleic acid dyes and aluminium oxide 

membrane filters for rapid epifluorescence 

enumeration of soil and sediment bacteria. Appl. 

Environ Microbiol, 64: 5000-5003. 

[26] http://rsbweb.nih.gov/ij/ 

[27] http://sites.google.com/site/cellcsoftware/ 

[28] ALMESJÖ L. & ROLFF C., 2007 - Automated 

measurements of filamentous cyanobacteria by 

digital image analysis. Limnol.Oceanogr. 

Methods, 5: 217-224. 

[29] KEPNER R.L. & PRATT J.R., 1994- Use of 

fluorochromes for direct enumeration of total 

bacteria in environmental samples: past and 

present. Microbiological, 58: 603-615. 

137


CHANGES IN BACTERIAL ABUNDANCE AND BIOMASS IN SANDY SEDIMENT 

MICROCOSMS SUPPLEMENTED WITH GASOLINE 

Dan Răzvan POPOVICIU 1 , Ioan ARDELEAN 1,2 

1 Ovidius University of Constanţa,Natural Sciences and Agricultural Sciences Faculty, Mamaia Avenue, no. 124, 

Constanţa, 900527, Romania, e-mail: dr_popoviciu@yahoo.com 

2 Biology Institute of Bucharest, Splaiul Independenţei, no. 296, 060031 Bucureşti, Romania, 

email:ioan.ardelean57@yahoo.com 

__________________________________________________________________________________________ 

Abstract: Bacterial abundance, biomass and morphological diversity were studied in three marine sediment 

microcosms: control, sediment supplemented with gasoline, and sediment supplemented with gasoline and 

ammonium nitrate. Microbial density (4.7-6.65 × 10 6 cells/cm 3 sediment in uncontaminated samples) and 

biomass (1.72-3.13 µg/cm 3 sediment) dropped significantly after gasoline addition. Ammonium nitrate favoured 

a faster recovery to initial values. Gasoline contamination also modified the proportion of bacterial morphotypes, 

increasing the percentage of rod-shaped cells. 

Keywords: Bacteria, microcosms, hydrocarbons, abundance, biomass, morphotypes, sandy sediments. 

__________________________________________________________________________________________ 


Hydrocarbon contamination is one of the most 

frequent and most dangerous forms of pollution 

affecting marine environments. Studying its effects on 

prokaryote communities is important, both 

theoretically and practically, opening the way to 

bioremediation. 

From a microbiological point of view, sediments 

and not the water column are the richest marine 

environment. Both sandy and muddy sediments show 

significant amounts of prokaryotes, playing a key role 

in the decomposition of organic matter and nutrient 

recycling.. 

Abundance, biomass and composition of 

sediment bacterial communities can be determined by 

many factors, such as the granulometric 

characteristics of the sediment, water dynamism, 

oxygenation, protozoan grazing etc [1, 2, 3]. 

Even though they cover a large part of the marine 

littoral enviroment, coastal sands are the less studied 

[1]. 

In order to evaluate the response of 

bacteriobenthos to various environmental changes, 

microcosms represent extremely valuable tools [4, 5]. 

The objective of the present study was to 

determine the effects of hydrocarbon addition on the 

abundance, biomass and morphological diversity of 

bacteria in marine sandy sediment microcosms. 


Microcosms. Sandy sediment was collected 

from the mediolittoral of a sandy beach in Constanţa, 

relatively close to the central headquarters of the 

“Ovidius” University, and wet sieved through a 2 mm 

sieve (in order to eliminate large particles and 

macrofauna) [6]. Three 1.4 L transparent plastic 

recipients were filled each with around 500 cm 3 of 

sand, covered by a 200 mL sea water column. All the 

microcosms were covered with transparent caps and 

stored at constant temperature (18°C) with 

illumination simulating the day-night cycle. 

The control microcosm was labeled “A”. 

Microcosm B was supplemented with 95 gasoline 

(1% final concentration). Microcosm C was 

supplemented with the same amount of gasoline, plus 

ammonium nitrate as a nutrient (0.005% final 

concentration). 

Sampling and fixation. Five samples consisting 

of sediment cores were collected from each 

microcosm at time intervals of 14 days. The first 

series of cores was taken just before the addition of 

gasoline and nutrient. 


Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) 

Sample collection was done using improvised 

piston corers (20 mL syringes with the forepart 

detached, but with the gradation intact). From each 

sample, the surficial 5 cm 3 (corresponding to a depth 

of 17.5 mm) was taken for analysis. 

Each sample was suspended in 5 ml of buffered 

formaline (4% final concentration) [7, 8, 9, 10]. The 

formaldehyde solution acts as a fixative, killing the 

microorganisms and preventing contamination and 

cell deformation. The labeled tubes containing the 

subsamples were preserved by refrigeration at +4°C. 

Cell separation. Dislodgement of bacteria 

attached to sand grains is an important step prior to 

analysis. The procedure used was adapted, with some 

modifications, from existing literature [11, 12, 13, 14, 

15]. 

Sediment suspensions were diluted 5-fold, 

incubated with Tween 80 (1 mg/mL final 

concentration) for 15 minutes and vortexed at 2 400 

r.p.m. for 5 minutes. 

Direct counting of bacteria. Microorganisms 

were visualised by epifluorescence microscopy, using 

3,6-dimethylaminoacridinic chloride (acridine 

orange) as a fluorochrome. This compound becomes 

highly fluorescent by binding to the nucleic acids, 

giving an orange-red fluorescence for single-stranded 

nucleic acids (mostly RNA) and a green one for 

double-stranded acids (DNA) [16]. Acridine orange 

stains both living and dead cells [17, 18]. 

The technique employed was an adapted and 

simplified version of the protocols used by other 

authors [3, 8, 10, 11, 19, 20]. 1 ml was collected from 

each suspension and incubated for 5 minutes with 1 

ml acridine orange (5 µg/mL final concentration). 

The resulting solution was filtered through a 0.45 µm 

Millipore filtering membrane, using a syringe and a 

Millipore holder. Filtered membranes were 

previously stained with Sudan Black, in order to 

reduce background fluorescence. 

Each filter was washed with 50-60 ml of distilled 

water, placed on a glass slide and examined using a 

Hund Wetzlar H 600 AFL 50 microscope, at an 

500× enlargement. An eyepiece grid micrometer was 

employed. 

For each filter, 15-20 grids were randomly 

chosen (from different areas of the membrane, except 

for its margins), photographed with a digital camera 

and visualised with MBF ImageJ for Microscopy 

140 

software (http://www.macbiophotonics.ca/downloads. 

htm.) [21]. 

Fluorescent cells in each grid were counted 

manually. Fluorescent anorganic particles and 

obviously eukaryotic structures (by size and 

morphology) were excluded. In case sediment 

particles masked bacterial cells, any bacteria found 

on the surface of such particles were counted twice 

[7, 8, 17, 22]. The mean bacterial density was 

calculated for each sample according to the following 

formula: 

N = n ×Af / Ag × V / v 

where: 

N = mean bacterial number per cm 3 of 

sediment; 

n = mean bacterial number per grid for each 

subsample; 

Ag = grid area; 

Af = filter area; 

v = volume of the filtered sediment 

suspension; 

V = volume of the total sediment suspension 

containing 1 cm 3 of sediment. 

Bacterial biomass estimation. All the 

microorganisms observed were classified into three 

morphological categories: cocci, bacilli (including 

coccobacilli and vibrios) and filamentous bacteria 

(those having a length more than five times greater 

than the width) [3]. Cell dimensions (diameter, 

respectively length and width) were measured using 

the grid micrometer. 

Biovolume was determined for each cell 

according to the formula [8, 16]: 

V = (π/ 4) d 2 (l – d / 3) 

where: 

l = cell length; 

d = cell width/diameter. 

For cocci, the formula becomes: 

V = πd 3 / 6 

To determine dry biomass based on the 

biovolume, several authors proposed different 

conversion factors. In the present study, the following 

formula was used [23]: 

md = 435 × V 0,86 

where: 

md = dry biomass (fg);

Dan Răzvan Popoviciu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010) 

V = cell volume (µm 3 ). 

Dry biomass was determined for each cell, 

calculating then the media for each sample. Total 

(wet) biomass can be approximated using a 

conventional mean value for bacterial cell density, of 

1.1 g/cm 3 [23, 24]. 


Bacterial cell abundance. The evolution of cell 

density in time (from 0 to 56 days) for each 

microcosm is shown in Fig. 1. 

Million cells 

7 

6.5 

6 

5.5 

5 

4.5 

4 

3.5 

3 

0 14 28 42 56 

Time (days) 

A 

B 

C 

Fig. 1. Number of bacterial cells (× 10 6 ) per cm 3 

of sediment. 

For undisturbed sediment cores, bacterial density 

ranged between 4.7-6.65 × 10 6 cells/cm 3 sediment 

with an average of 5.52 × 10 6 cells/cm 3 . 

These values are within the variation limits of 

littoral sediment microbial density (although data 

found in literature is distributed over a wide range). 

For comparison, here are some bacterial densities: 10 9 

cells/g dry sand [20], 5 × 10 8 -1.5 × 10 9 cells/g 

sediment [9] and 7-9 × 10 7 cells/g [25] on the U.S.A. 

East Coast, 1.91-7.32 × 10 7 cells/g dry sediment, in 

Eastern Canada, at the waterline [1], 3.6 × 10 8 cells/g 

dry sediment, in Florida [26], 6.8-20.3 × 10 8 

141 

cells/cm 3 , fine sands in a Mexican tropical lagoon, 1.2 

m depth [27], 7 × 10 8 -6.7 × 10 9 cells/cm 3 , Baltic Sea 

[28], over 5.12 × 10 8 cells/g dry sediment, Western 

Mediterranean Sea [29], 1.5 × 10 8 cells/g dry sand 

[10], 6-8 × 10 9 cells/g sediment [30] and 3.54-8.08 × 

10 9 cells/g [3] in the Adriatic Sea, at several meters 

depth, 0.2-1 × 10 9 cells/g dry sediment, in littoral 

sands in the Gulf of Tokyo [14], 2.56-4.46 × 10 6 

cells/g, at 2 m depth, in North Sea [31]. 

The addition of gasoline caused a decrease in cell 

abundance to values as low as 3.6 × 10 6 cells/cm 3 . A 

return to densities similar to the initial ones was 

observed in the last samples. The recovery was faster 

in the microcosm supplemented with ammonium 

nitrate (28 days). 

Direct cuantification of bacteria through 

epifluorescence microscopy has some limitations. 

Cell masking by sediment particles, background 

fluorescence, lack of an efficient method to 

distinguish prokaryotes from eukaryotes, the poor 

quality of some photographs etc., can cause 

overestimation or underestimation of real abundance 

[8, 17, 22]. The method used for bacterial dispersion 

from sediment grains can also influence the results 

[9]. 

An important factor that can cause 

underestimation of bacterial abundance is the 

extremely small size of some microorganisms. Many 

bacteria have diameters below 0.3 microns, and can 

be very difficult or even impossible to visualise, 

depending on the optical means employed. Some of 

them can even pass through usual filtering 

membranes. According to some authors such 

ultramicrobacteria constitute up to 72% of the soil 

microbiota, and it seems that they have similar 

proportions in marine environments [17]. In 

conclusion, all data obtained using direct counts 

should be regarded as relative. 

It should be noted that not all the bacteria 

ennumerated with acridine orange are alive. Living 

bacteria constitute usually less than one third, rarely 

reaching 60% of the total number. The rest are dead 

cells, or even cell fragments [10, 32].


Biomass (µg) 

3.5 

3 

2.5 

2 

1.5 

1 

0 14 28 42 56 

Time (days) 

Fig. 2. Bacterial dry biomass (µg/cm 3 ). 

Bacterial biomass 

Biomass showed large variations, from 1.36 to 

3.13 µg/cm 3 sediment (equivalent to 4.3 to 11.4 µg 

total biomass/cm 3 ). On average, the highest biomass 

was determined for the undisturbed sediment (an 

average of 2.27 µg/cm 3 ). The addition of gasoline was 

followed by a decrease in microcosms B and C. The 

average value for contaminated sediment in 

microcosm B was only 1.7 µg/cm 3 , while in C, it was 

higher (2.12 µg/cm 3 ), showing a faster recovery. 

The importance of nitrogenous nutrients in the 

recovery of natural microbiota after hydrocarbon 

pollution is consistent with data in existing literature 

[33, 34]. 

The exact determination of bacterial biomass can 

be affected by various technical and mathematical 

factors. Different fluorochromes can give different 

results [16]. The selected biovolume to biomass 

conversion factor influences the final results. Also, it 

was demonstrated that coastal marine sediments 

contain significant numbers of disk-shaped bacteria 

and counting them as cocci would overestimate their 

volume [35]. 

Proportion of major bacterial morphotypes. 

As specified above, bacteria were classified into three 

groups: cocci, rods and filamentous. Their proportion 

in the total abundance, for each microcosm and 

collection time is shown in Fig. 3 (a,b,c). 

A 

B 

C 

142 

100% 

80% 

60% 

40% 

20% 

0% 

100% 

80% 

60% 

40% 

20% 

0% 

100% 

80% 

60% 

40% 

20% 

0% 

Microcosm A 

0 14 28 42 56 

Time (days) 

Microcosm B 

0 14 28 42 56 

Time (days) 

Microcosm C 

0 14 28 42 56 

Time (days) 

Filamentous 

Rods 

Cocci 

Filamentous 

Rods 

Cocci 

Filamentous 

Rods 

Cocci 

Fig.3 (a,b,c). Percentage of major bacterial 

morphotypes


Most of the bacterial cells (72-92%) observed 

were spherical (in accordance to data obtained in 

a) 

a) 

b) 

c) 

Fig. 4. Main bacterial morphotypes: a) cocci; b) 

rods; c) filamentous (bar = 10 µm) 

143 

marine sediments by Šestanović et al. [3] and 

Popoviciu [36]). 

The proportion of rod-shaped bacteria (including 

coccobacilli and vibrios) was different among the 

three microcosms. In undisturbed sediment, their 

percentage was generally below 15% (note: in 

microcosm A, at T3, the high percentage was due to a 

single large colony of small rods), with an average of 

13.9%. In gasoline contaminated sediment, rodshaped 

bacteria constituted a larger part of the 

microbiota, with an average of 23.4%. The 

proportion of filamentous bacteria was insignificant. 

Bacterial assemblages were rare, in concordance 

to the observations made by Novitsky & MacSween 

[1]. 

It should be noted that classification of small 

bacteria (cells with diameters below 0.6 microns 

formed the majority) into morphotypes is prone to 

errors. This is due to the fluorescent halo that appears 

around cells, causing very small sized bacilli or 

vibrios to be counted as cocci [19]. 


Hydrocarbon contamination affects marine 

sediment microbiota in terms of abundance, biomass 

and composition. 

Addition of nitrogenous nutrients (ammonium 

nitrate) favours a faster recovery to initial parameters. 

Epifluorescence microscopy is a useful tool for 

evaluating the reaction of sediment bacteria to 

environmental changes. In perspective, use of 

differential fluorochromes and correlation to 

cultivation techniques are to be employed in such 

studies. 

5. References 

[1] NOVITSKY, J.A., MACSWEEN, M.C., 1989 – 

Microbiology of a high energy beach sediment: 

evidence for an active and growing community. 

Mar. Ecol. Prog. Ser. 52: 71-75. 

[2] BRUNE, A., FRENZEL, P., CYPIONKA, H., 

2000 – Life at the oxic-anoxic interface: 

microbial activities and adaptations. FEMS 

Microbiol. Rev. 24: 691-710.


[3] ŠESTANOVIĆ, S., SOLIĆ, M., 

KRUSTULOVIĆ, N., BOGNER, D., 2005 – 

Volume, abundance and biomass of sediment 

bacteria in the eastern mid Adriatic Sea. Acta 

Adriat. 46: 177-191. 

[4] RÖLING, W.F.M., MILNER, M.G., JONES, 

D.M., LEE, K., DANIEL, F., SWANNELL, 

R.J.P., HEAD, I.M., 2002 – Robust 

hydrocarbon degradation and dynamics of 

bacterial communities during nutrient-enhanced 

oil spill bioremediation. Appl. Environ. 

Microbiol. 68: p. 5537-5548. 

[5] MIRALLES, G., NÉRINI, D., MANTÉ, C., 

ACQUAVIVA, M., DOUMENQ, P., 

MICHOTEY, V., NAZARET, S., BERTRAND, 

J.C., CUNY, P., 2007 – Effects of spilled oil on 

bacterial communities of Mediterranean coastal 

anoxic sediments chronically subjected to oil 

hydrocarbon contamination. Microb. Ecol. 54: 

646-661. 

[6] FISH, K.M., PRINCIPE, J.M. 1994 – 

Biotransformations of Aroclor 1242 in Hudson 

River test tube microcosms. Appl. Environ. 

Microbiol. 60: 4289-4296. 

[7] SCHALLENBERG, M., KALFF, J., 

RASMUSSEN, J.B., 1989 – Solutions to 

problems in enumerating sediment bacteria by 

direct counts. Appl. Environ. Microbiol. 55: 

1214-1219. 

[8] FRY, J.C., 1990 – Direct methods and biomass 

estimation. Meth. Microbiol. 22: 41-85. 

[9] EPSTEIN, S.S., ALEXANDER, D., COSMAN, 

K., DOMPÉ, A., GALLAGHER, S., 

JARSOBSKI, J., LANING, E., MARTINEZ, 

R., PANASIK, G., PELUSO, C., RUNDE, R., 

TIMMER, E., 1997 – Enumeration of sandy 

sediment bacteria: Are the counts quantitative or 

relative? Mar. Ecol. Prog. Ser. 151: 11-16. 

[10] LUNA, G.M., MANINI, E., DANOVARO, R., 

2002 – Large fraction of dead and inactive 

bacteria in coastal marine sediments: 

comparison of protocols for determination and 

ecological significance. Appl. Environ. 

Microbiol. 68: 3509-3513. 

[11] FALLON, R.D., NEWELL, S.Y., 

HOPKINSON, C.S., 1983 – Bacterial 

production in marine sediments: will cell- 

144 

specific measures agree with whole-system 

metabolism? Mar. Ecol. Prog. Ser. 11: 119-127. 

[12] ELLERY, W.N., SCHLEYER, M.H., 1984 – 

Comparison of homogenization and 

ultrasonication as techniques in extracting 

attached sedimentary bacteria. Mar. Ecol. Prog. 

Ser. 15: 247-250. 

[13] EPSTEIN, S.S., ROSSEL, J., 1995 – 

Enumeration of sandy sediment bacteria: search 

for optimal protocol. Mar. Ecol. Prog. Ser 117: 

289-298. 

[14] KUWAE, T., HOSOKAWA, Y., 1999 – 

Determination of abundance and biovolume of 

bacteria in sediments by dual staining with 4’,6diamidino-2-phenylindole 

and acridine orange: 

relationship to dispersion treatment and 

sediment characteristics. Appl. Environ. 

Microbiol. 65: 3407-3412. 

[15] BENNETT, P.C., ENGEL, A.S., ROBERTS, 

J.A., 2006 – Counting and imaging bacteria on 

mineral surfaces. in Patricia, J., Maurice, A., 

Warren, L.A. (eds.). Methods of Investigating 

Microbial-Mineral Interactions. CMS Workshop 

Lectures, Vol. 14: 37-78, The Clay Mineral 

Society, Chantilly. 

[16] SHERR, B., SHERR, E., DEL GIORGIO, P., 

2001 – Enumeration of total and highly active 

bacteria. Meth. Microbiol. 30: 129-159. 

[17] KEPNER, R.L., PRATT, J.R., 1994 – Use of 

fluorochromes for direct enumeration of total 

bacteria in environmental samples: past and 

present. Microbiol. Rev. 58: 603-615. 

[18] MCFETERS, G.A., YU, F.P., PYLE, B.H., 

STEWART, P.S., 1995 – Physiological 

assessment of bacteria using fluorochromes. J. 

Microbiol. Meth. 21: 1-13. 

[19] WATSON, S.W., NOVITSKY, T.J., QUINBY, 

H.L., VALOIS, F.W., 1977 – Determination of 

bacterial number and biomass in the marine 

environment. Appl. Environ. Microbiol. 33: 

940-946. 

[20] MONTAGNA, P.A., 1982 – Sampling design 

and enumeration statistics for bacteria extracted 

from marine sediments. Appl. Environ. 

Microbiol. 43: 1366-1372. 

[21] COLLINS, T.J., 2007 – ImageJ for microscopy. 

BioTechniques 43: 25-30.


[22] GOUGH, H.L., STAHL, D.A., 2003 – 

Optimization of direct cell counting in sediment. 

J. Microbiol. Meth. 52: 39-46. 

[23] LOFERER-KRÖßBACHER, M., KLIMA, J., 

PSENNER, R., 1998 – Determination of 

bacterial cell dry mass by transmission electron 

microscopy and densitometric image analysis. 

Appl. Environ. Microbiol. 64: 688-694. 

[24] BAKKEN, L.R., OLSEN, R.A., 1983 – Buoyant 

densities and dry-matter contents of 

microorganisms: conversion of a measured 

biovolume into biomass. Appl. Environ. 

Microbiol. 45: 1188-1195. 

[25] HYMEL, S.N., PLANTE, C.J., 1998 – 

Improved method of bacterial enumeration in 

sandy and deposit-feeder gut sediments using 

the fluorescent stain 4,6-diamidino-2phenylindole 

(DAPI). Mar. Ecol. Prog. Ser. 

173: 299-304. 

[26] PROCTOR, L.M., SOUZA, A.C., 2001 – 

Method for enumeration of 5-cyano-3,2-ditoyl 

tetrazolium chloride (CTC)- active cells and 

cell-specific CTC activity of benthic bacteria in 

riverine, estuarine and coastal sediments. J. 

Microbiol. Meth. 43: 213-222. 

[27] FERRARA-GUERRERO, M.J., 

CASTELLANOS-PAÉZ, M.E., GARZA- 

MOURIÑO, G., 2007 – Variation of a benthic 

heterotrophic bacteria community with different 

respiratory metabolisms in Coyuca de Benitez 

coastal lagoon (Guerrero, Mexico). Rev. Biol. 

Trop. (Int. J. Trop. Biol.) 55: 157-169. 

[28] DIETRICH, D., ARNDT, H., 2000 – Biomass 

partitioning of benthic microbes in a Baltic 

inlet: relationships between bacteria, algae, 

heterotrophic flagellates and ciliates. Mar. Biol. 

136: 309-322. 

[29] DANOVARO, R., FABIANO, M., BOYER, 

M., 1994 – Seasonal changes of benthic bacteria 

in a seagrass bed (Posidonia oceanica) of the 

Ligurian Sea in relation to origin, composition 

and fate of the sediment organic matter. Mar. 

Biol. 119: 489-500. 

[30] PUSCEDDU, A., FIORDELMONDO, C., 

DANOVARO, R., 2005 – Sediment 

resuspension effects on the benthic microbial 

loop in experimental microcosms. Microb. Ecol. 

50: 602-613. 

145 

[31] LUNAU, M., LEMKE, A., WALTHER, K., 

2005 – An improved method for counting 

bacteria from sediments and turbid 

environments by epifluorescence microscopy. 

Environ. Microbiol. 7: 961-968. 

[32] ZWEIFEL, U.L., HAGSTRÖM, Å., 1995 – 

Total counts of marine bacteria include a large 

fraction of non-nucleoid-containing bacteria 

(ghosts). Appl. Environ. Microbiol. 61: 2180- 

2185. 

[33] HIGASHIHARA, T., SATO, A., SIMIDU, U., 

1978 – An MPN method for the enumeration of 

marine hydrocarbon degrading bacteria. Bull. 

Japan. Soc. Sci. Fish. 44: 1127-1134. 

[34] HAZEN, T.C., 2010 – Biostimulation, in 

Timmis, K.N. (ed.). Handbook of Hydrocarbon 

and Lipid Microbiology, 4517-4530, Springer- 

Verlag Berlin Heidelberg. 

[35] MUDRYK, Z.J., PODGÓRSKA, B., 2006 – 

Scanning electron microscopy investigation of 

bacterial colonization of marine beach sand 

grains. Baltic Coastal Zone 10: 61-72. 

[36] POPOVICIU, D.R., 2009 – Aspecte cantitative 

şi morfo-structurale ale microbiotei din 

sedimente marine nisipoase de la litoralul 

românesc al Mării Negre. Master’s thesis, 

Ovidius University of Constanţa, Faculty of 

Natural Sciences and Agricultural Sciences.


THE FORMATION OF BACTERIAL BIOFILMS ON THE HYDROPHILE 

SURFACE OF GLASS IN LABORATORY STATIC CONDITIONS: THE EFFECT 

OF TEMPERATURE AND SALINITY 

Aurelia Manuela MOLDOVEANU *, Ioan I. ARDELEAN ** 

* Ovidius” University Constanta, 1Universitatii Alley, Building B, 900527 Constanta, Romania, 

aurelia.moldoveanu@yahoo.com 

** Biology Institute of Bucharest, 296 Splaiul Independenţei, 060031 Bucharest, Romania, 

ioan.ardelean57@yahoo.com 

__________________________________________________________________________________________ 

Abstract: In the case of temperature variation, at 18ºC there is an increase of the cellular density from 

12∙10 2 cel/mm 2 to 62∙10 2 cel/mm 2 , while at 6 ºC cellular density increases from 5∙10 2 cel/mm 2 to 55∙10 2 

cel/mm 2 . The results obtained show that cellular density in the case of biofilms formed at 6 ºC is lower 

compared to cellular density of biofilms formed at 18 ºC. Salinity modification from 15g/l to 10g/l 

determined an increase of cellular density from 4∙10 2 cel/mm 2 to 54∙10 2 cel/mm 2 , while the modifications 

of the osmotic conditions in the marine environment due to salinity decrease to 5g/l led to an increase of 

the cellular density from 2∙10 2 cel/mm 2 to 49∙10 2 cel/mm 2 . The variation of temperature and salinity of 

seawater in “in vitro” conditions influenced the process of bacterial adherence and formation of the initial 

layers of the biofilms by the modification of the density of the adherent cells. 

Keywords: quorum sensing, exopolysaccharides, matrix, microecosystem, microfouling. 

__________________________________________________________________________________________ 


Biofilms are complex structures made up of 

cells and exopolysaccharides which form at the level 

of interfaces and which are intensely studied because 

of their fundamental importance and applicability in 

the environmental domain, biotechnology and 

medicine [1,2,3,4,5]. 

Marine bacteria form biofilms in “in situ” 

conditions, under the influence of various 

environmental factors. Hydrostatic pressure, solar 

radiation, temperature, salinity, pH, oxidation 

potential and nutrients existing on the surfaces are 

physic-chemical factors that influence the activity of 

microorganisms, but their role on the marine bacterial 

populations is still being studied [6,7,5]. Among 

these factors, temperature and salinity have major 

importance for all living organisms, especially for 

those in the marine environment, where 

microorganisms are subjected to extremely wide 

variations which allowed them to survive from the 

beginning of life on Earth. They are the only 

organisms that can adapt to extreme environments 

[8,9,10]. 

In laboratory conditions, the variation of 

environmental factors is essential in the formation of 

biofilms. It is important to know which factor has the 

most influence on the adherent bacterial cells. Thus, 

the growth and multiplication of microorganisms is 

the result of a number of coordinated metabolic 

reactions whose normal development is ensured by an 

optimal temperature [11,12,13]. 

The marine bacteria in the structure of biofilms 

react to temperature and modify their bacterial 

metabolism and the mechanism for the regulation of 

genes depending on how this factor varies, most 

species being studied between their optimal 

temperature limits due to the mesophile character 

[14]. Thus, an increase by 10 ºC of the initial 

temperature determines an increase of the speed of 

the chemical reactions and gene regulation 

mechanism. Consequently, the speed of the enzymatic 

processes increases progressively as the temperature 


The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) 

rises until it reaches the optimum level and then the 

speed decreases progressively [15]. 

The natural environments offer microorganisms 

different conditions of salinity, from very low 

concentrations (rivers and lakes) to the very high 

concentrations of salty lakes and seas, or even to 

those that represent true saturated solutions. Thus, 

salinity becomes a variable factor in the marine 

environment which is important for the 

microorganisms within the biofilms as they are 

influenced by it according to the degree of tolerance 

to the concentration of NaCl and the mechanism for 

the regulation of the available ions [16]. The study of 

the effect of temperature and salinity on the temporal 

dynamics of bacterial cell density on the hydrophile 

surface of glass in laboratory static conditions leads 

to personal data regarding the initial stages of biofilm 

formation. 


In our experiments, we used two static methods 

in order to determine the environmental factors with 

role in biofilm formation: the Henrici method 

[17,18,19], widely employed in the study of 

adherence and the microbial fishing method [20,21], 

a more recent adaptation of the classical method. 

The surfaces were subjected to a sterilization 

process in order to diminish the possible 

contamination with microorganisms of the glass 

slides which will serve as support for the adherent 

marine bacteria. The slides were degreased with 

ethanol 70% and sterilized in the drying oven at 180 

ºC for one hour [22]. 

In order to obtain biofilms, two types of liquid 

culture media were used: seawater from the littoral 

zone and seawater kept in aquarium conditions in the 

Laboratory for Biodiversity Investigation within 

“Ovidius” University of Constanta. The aquarium 

seawater is frequently used in the study of biofilms 

and marine microfouling and it was used in order to 

observe the possible facilitation of their formation 

[23]. 

The method used is accomplished in static 

conditions in sterile containers in which 100 ml of 

seawater were poured and the slides were introduced. 

This type of method is more advantageous for the 

formation of biofilms when there is no system for 

148 

water recirculation, according to [24], who claims 

that the methods with continuous flux prevent the 

rapid formation of biofilms within the first hours. 

The support slides for the adherent bacteria 

were positioned according to the mentioned methods 

in an inclined position compared to the classical 

method, in order to avoid the sedimentation 

phenomenon which determines the occurrence of 

high densities of the adherent marine bacteria [25]. 

The experiment was accomplished in a 

thermostatic room at a constant temperature of 18 ºC 

in the Laboratory for Biodiversity Investigation 

within “Ovidius” University of Constanta and in a 

refrigerator at a constant temperature of 6 ºC. The 

salinity modification was done only for the littoral 

seawater and not for the aquarium water which is a 

microecosystem. Thus, seawater salinity, which has a 

normal value of 15g/l was modified by adding 

osmosis water and certain mixtures per liter obtaining 

thus two experimental versions: in the first version, 

normal salinity was decreased to 10g/l by adding 

333ml of osmosis water in 666ml of seawater; in the 

second version a salinity of 5g/l was obtained by 

adding 666 ml of osmosis water in 333 ml of 

seawater. 

The study of biofilms was accomplished over a 

period of 36 hours during which there was an interval 

when no samples were collected. Sample collection 

occurred for 12 hours in the first day hourly, followed 

by an interval of 12 hours when no samples were 

collected and again the following day samples were 

collected every two hours for 12 hours. 

After collection the slides were subjected to a 

process of fixation with 2.5% formaldehyde solution 

in artificial seawater (solution with marine salts with 

a concentration of 18g/l, similar to the Black Sea) for 

30 minutes and then subjected to desalinization by 

washing for 10 minutes in three successive solutions 

with the following content: 75% artificial seawater 

with 25% osmosis water, 50% artificial seawater with 

50% osmosis water and 100% osmosis water. The 

desalinization was realized in order to prevent the 

formation of salt crystals which absorb the 

fluorescent coloring matter and reflect it, affecting 

thus the cell visualization [26]. 

After desalinization, the samples were 

introduced in a solution with 0.5% gentian violet in 

10 ml ethanol and 90 ml distilled water for one

Aurelia Manuela Moldoveanu, Ioan Ardelean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010) 

minute. Afterwards, they were abundantly washed 

twice with osmosis water in order to eliminate the 

excess of coloring matter [27]. 

The sample investigation was done by means of 

the Hund microscope, cell counting being done using 

an ocular grid, calibrated according to the standard 

procedure [28]; cells within 20 microscopic fields 

per each sample were counted, according to the 

standard counting procedures for the surface bacteria 

[29]. Thus, the values of cellular density are 

expressed on the graphs represented by the mean of 

the 80 microscopic fields per each sample. 


3.1 The chemical analysis of water 

There are differences between the two types of 

culture media used for the generation of bacterial 

biofilms on the hydrophile surface of glass slides and 

in order to emphasize their existence we analyzed the 

seawater samples in the Chemistry Laboratory within 

the “Grigore Antipa” Marine Research Institute of 

Constanta (Table 1). 

The chemical analysis of seawater emphasized 

the existence of differences among the chemical 

parameters: salinity, pH, concentration of inorganic 

substances between the two types of seawater used. 

The littoral seawater has normal parameters also 

registered in previous years [30], but the aquarium 

seawater has values well over the normal limit with 

an increase of over 10g/l of salinity and a decrease of 

pH from 8.12 (normal value for littoral seawater) to 

6.56 units for the aquarium water, almost two units 

less than the initial value. The concentration of 

inorganic substances is well above the normal one for 

seawater. The concentration of nitrates is three times 

higher compared to the normal value, while the 

concentration of polyphosphates is over 84 times 

higher. 

The existence of these differences between the 

two used culture media can cause changes in the 

formation manner of bacterial biofilms in liquid 

medium, as well as the temporal dynamic of their 

formation. The bacterial biofilms formed are an 

assemblage of surface-associated microbial cells that 

149 

is enclosed in an extracellular polymeric substance 

matrix. 

3.2 The formation of biofilms under the 

influence of temperature 

Figure one shows the values of cellular density 

obtained after the modification of the temperature 

factor for the biofilms formed on the hydrophile 

surface of glass slides and collected from the 

containers with littoral seawater kept at a constant 

temperature of 18ºC and 6 ºC, respectively. 

The data analysis emphasized the existence of 

successive stages for the formation of biofilms. Thus, 

in the case of the biofilms formed at 18 ºC, one hour 

after the slides immersion the cellular density is 

12∙10 2 cel/mm 2 . This value doubles eight hours later 

to 25 ∙10 2 cel/mm 2 and increases progressively to a 

tendency to triple the cellular density to 37 ∙ 10 2 

cel/mm 2 11 hours later. After 12 hours, during which 

the slides were left over night, the following day the 

cellular density reaches the value of 45∙10 2 cel/mm 2 

24 hours after immersion. The value increases 

progressively to 60∙10 2 cel/mm 2 36 hours after 

immersion 

For the seawater in the containers kept at 6 ºC in 

the refrigerator, there is a progressive increase from 5 

∙10 2 cel/mm 2 only one hour after immersion and a 

doubling of this value seven hours later to 10 ∙10 2 

cel/mm 2 , as well as tripling to 15∙10 2 cel/mm 2 eight 

hours later. The following day, after 12 hours, the 

cellular density was 41∙10 2 cel/mm 2 and increased 

progressively to 54∙10 2 cel/mm 2 . 

The progression of cellular density growth is 

over 2.3 for the biofilms formed at 18 ºC during the 

first 12 hours and below 1.2 after 24 hours. Also, in 

the case of the biofilms formed in containers kept at 6 

ºC, the progression is over 1.9 during the first 12 

hours and below 1.1 after 24 hours. On the first day, 

after 12 hours, there is a difference of approx. 9∙10 2 

cel/mm 2 between the two progressions of density 

growth, depending on temperature. 24 hours later, the 

difference is below 8∙10 2 cel/mm 2 and 36 hours later 

it rises to 10∙10 2 cel/mm 2 . 

A number of experiments regarding bacterial 

adhesion were accomplished on different types of 

surfaces (copper, PVC and polybuten) by Rogers [31] 

at different temperatures (20 ºC, 40 ºC, 50 ºC and 60


ºC) using the species Legionella pneumophila (a 

species with wide temperature limits between 5.7 and 

63 ºC) and other strains of non-Legionella type over a 

period of 21 days. As a result, it was observed that 

the used strains displayed a logarithmic growth with a 

density value of 1.3∙10 4 cel/cm 2 in the growing phase 

and 7.56∙10 4 cel/cm 2 on polybutylene and PVC 

surfaces for the non-Legionella strains at 20 ºC and 

4.25∙10 4 cel/cm 2 for polybutylene surface at 60 ºC. It 

is evident that the colonization is higher on the 

hydrophobe surfaces at 20 ºC, compared to 60 ºC 

when the number of bacteria decreases due to the 

exceeding of the optimal temperature for 

microorganism development. 

Experiments regarding the colonization of 

surfaces by the bacterium Bdellovibrio bacteriovorus 

were accomplished by Kelley [32] under the 

influence of different temperatures (between 4 and 29 

ºC), using clam valves, glass and polystyrene as 

substrate, and observing the existence of positive 

correlations in the case of the factor temperature and 

the formation of biofilms, with maximum association 

of cells in the biofilms at 18 ºC and a minimum one at 

14 ºC, as well as a significant decrease of density at 

temperatures below 5 ºC after 24 hours, followed by 

a progressive increase of density 120 hours after the 

beginning of the experiment. 

The values obtained demonstrated a logarithmic 

increase of the number of adherent cells from 1.1 ∙10 5 

CFU/cm 2 to 1.4∙10 5 CFU/cm 2 for the clam valves, 

1.7∙10 3 CFU/cm 2 and 1.8∙10 4 CFU/cm 2 for glass 

and 5.4∙10 3 CFU/cm 2 and 1.0 ∙10 4 CFU/cm 2 for 

polystyrene. 

A number of experiments regarding the capacity 

of accomplished certain isolates of 

Stenotrophomonas maltophilia to form biofilms in 

variable temperature conditions (18 ºC, 32 ºC, 37 ºC) 

by were realized by Di Bonaventura [33] using 

different strains. There is an increase of the quantity 

of biofilms for the strains exposed to 32 ºC after one 

day to 0.680 BPI compared to those exposed to 18 ºC 

(0.557 BPI) and 37 ºC (0.491 BPI). In what regards 

the used strains, the temperature did not modify 

significantly their distribution: 82% of those used 

formed biofilms and only 2% did not form them. One 

strain formed biofilms only at 18 ºC and two strains 

only at 32 ºC. The capacity to forms biofilms is 

150 

important even at room temperature (18 ºC), but the 

adherence value is lower. 

The following day, after the 12 hour interval 

when no samples were collected, the density value 

was 41∙10 2 cel/mm 2 and there was a progressive 

growth towards 55∙10 2 cel/mm 2 . 

Data in specialized literature confirm the 

existence of a growth in bacterial density depending 

on the exposure time of the surfaces to aquatic 

environment and the increase of temperature. Thus, at 

18 ºC, up to 25-30 ºC, there is an optimal bacterial 

growth. But temperatures over 35 ºC, 40 ºC and 50 

ºC affect the formation of biofilms because the 

optimum limits for the survival of certain bacterial 

species are exceeded 

Our experiments took place between the 

optimum limits for mesophile bacteria, noticing an 

increase of the density values at 18 ºC, compared to 6 

ºC (kept in a refrigerator). 

In the case of the slides immersed in containers 

with aquarium water at 18 ºC, Figure 2 displays an 

increase of the bacterial density from 16∙10 2 cel/mm 2 

one hour after immersion to a double value of 32∙10 2 

cel/mm 2 eight hours later. After 12 hours, during 

which the slides were left over night, there is a 

tendency for the tripling of the cellular density to 

49∙10 2 cel/mm 2 22 hours after the immersion of the 

slides into liquid medium and a progressive increase 

towards 62∙10 2 cel/mm 2 . 

For the containers kept at 6 ºC, the density 

increases to 5∙10 2 cel/mm 2 one hour from immersion 

towards a double value of 13∙10 2 cel/mm 2 seven 

hours later and a progressive growth from 22 ∙10 2 

cel/mm 2 ten hours later when there is a tendency for 

a triple value of the density of adherent bacteria. 

The growth occurs based on a progression of 

2.5 in the case of biofilms formed in aquarium water 

kept at 18 ºC during the first 12 hours. There is a 

decrease to 1.1 after 24 hours from immersion. The 

difference between the two progressions is 4∙10 2 

cel/mm 2 during the first 12 hours from immersion 

and it increases to 8 ∙10 2 cel/mm 2 24 hours later. It 

decreases 36 hours later to 7 ∙10 2 cel/mm 2 . 

In variable conditions of temperature, the 

bacterial colonization occurs more quickly in 

aquarium seawater. Thus, Toren [34] realizes 

experiments regarding the formation of biofilms 

(Vibrio sp. strain AK-1) on a coral surface in case of


temperature variation (16º C, 23º C, 29º C) and 

registers a decrease of the quantity of inoculate from 

1.2 ∙10 8 cel/l to 1.2∙10 2 cel/l used with the increase of 

temperature, as well as an increased adhesion at high 

temperatures during the first hours from immersion. 

Experiments were realizes by Else [5] regarding 

the bacterial colonization of the hydrophile surface of 

metals (stainless steel, titanium and nickel) in 

variable conditions of temperature (30º C, 60º C and 

70º C) and humidity over a longer period of time 

(from a few days to 18 months). They observed an 

increase of adherent bacteria between 1.06∙10 2 cel/cm 

2 and 7.61 ∙10 2 cel/cm 2 at a temperature of 30º C on 

steel plates. They also observed a decrease of the 

number of bacteria from the first day for the plates 

exposed to high temperatures (60º C and 70º C), 

especially on those of nickel and steel. 

In what regards the role of the bacterial film in 

the mediation of invertebrate attachment and fouling 

formation, Lau et al. [35] realized experiments at 

different temperatures (16º C, 23º C and 30º C), 

noticing an increase in the number of bacteria from 

14.3∙10 3 cel/mm -2 at 16º C to 21.2∙10 3 cel/mm -2 at 

30º C. The experiments emphasized a more 

significant influence of the temperature on the 

biomass than on the bacterial density. 

A number of experiments regarding the 

formation of biofilms in different conditions of 

temperature were realized by Di Bonaventura [36] 

together with other collaborators accomplish in 2007 

(4º C, 12º C, 22º C, 37º C) by Listeria 

monocytogenes on the hydrophile surface of glass, 

steel and the hydrophobe surface of polystyrene. The 

results emphasized a progressive increase on the 

surface at 4 ºC of 0.206, at 12 ºC BPI to 0.233 BPI, 

22º C to 0.366 BPI, in comparison to polystyrene and 

stainless steel. At 37º C the values are close to those 

from the three surfaces studied, but there is also 

greater species variability. Still, the most 

considerable growth of 1.275 was obtained on the 

hydrophobe surface of polystyrene. 

Bacterial density registers an increase of the 

adherent bacteria with higher values for the biofilm 

formed in aquarium water kept at 18 ºC, compared to 

the one kept at 6 ºC. The values obtained are higher 

than those for seawater, which is due to the different 

physical and chemical properties of aquarium water 

and to the nutrients. Adherent marine bacteria attach 

151 

themselves to surfaces and form microcolonies in the 

first hour after immersion in the marine medium. 

They grow in size with the immersion period, data 

confirmed by [24]. 

3.3 The formation of biofilms under the 

influence of salinity 

Variation of salinity was done in order to 

observe the influence of osmotic conditions on the 

process of bacterial adherence and the formation of 

the initial phases of biofilms. For the slides immersed 

in containers with seawater with 15g/l salinity, Figure 

3 displays an increase of bacterial density to 12∙10 2 

cel/mm 2 one hour after immersion towards a 

doubling of this value to 25∙10 2 cel/mm 2 eight hours 

later. 

The slides were left over night for 12 hours and 

the following day there was a progressive increase of 

the cellular density value of 49∙10 2 cel/mm 2 where 

there is a tendency for a triple value towards 62∙10 2 

cel/mm 2 . 

In the case of containers with seawater with 

modified salinity (addition of osmosis water 10g/l), 

the bacterial density increased to 4∙10 2 cel/mm 2 one 

hour after immersion to a double value of 8∙10 2 

cel/mm 2 after four hours and the progressive increase 

from 18 ∙10 2 cel/mm 2 after eight hours when there is 

a tendency to triple the value of bacterial density. 

After the 12 hour interval when the slides were left 

over night in containers, there is an increase of the 

cellular density from 41∙10 2 cel/mm 2 24 hours after 

immersion to 54∙10 2 cel/mm 2 36 hours after 

immersion. The growth progression during the first 

12 hours in the case of the biofilms formed at a 

salinity of 15g/l is higher, with a value of 2.3 and 

displays a decrease after 24 hours to 1.1. In the case 

of the biofilms formed at a salinity of 10g/l, the 

growth progression is 2.4 during the first 12 hours 

and it decreases after 24 hours to 1.1. The difference 

between the two progressions is 2∙10 2 cel/mm 2 in the 

first 12 hours, it increases to 8∙10 2 cel/mm 2 24 hours 

after immersion and remains constant at this value 

until 36 hours. 

In the case of containers with seawater with 

modified salinity (addition of osmosis water 5g/l), 

Figure 4 displays an increase of bacterial density 

from 2∙10 2 cel/mm 2 one hour after immersion to a


double value of 4∙10 2 cel/mm 2 three hours later and 

the progressive increase to 9 ∙10 2 cel/mm 2 six hours 

later when the tendency is for a triple value of the 

bacterial density. The slides collected after 12 hours 

display a progressive increase of cellular density from 

31∙10 2 cel/mm 2 24 hours after immersion to 49∙10 2 

cel/mm 2 36 hours later. 

The increase is accomplished based on a 

progression with a value of 2.3 in the first 12 hours in 

the case of the biofilms formed at 15g/l salinity and a 

decrease of this value to 1.1 after 24 hours. 

For the biofilms formed at 5g/l salinity, the 

value of the growth progression is 1.5 in the firs 12 

hours, which drops to 1.4 in the following 24 hours. 

Between the two growth progressions there are 

differences between the values of cellular density. 

Thus, after 12 hours, the difference is 13∙10 2 cel/mm 

2 and it drops after 24 hours to 8∙10 2 cel/mm 2 , but 

increases after 36 hours to 13∙10 2 cel/mm 2 . 

While studying the colonization of surfaces by 

the bacterium Bdellovibrio bacteriovorus, [32] 

realized experiments under the influence of different 

temperatures and observed the existence of a 

colonization tendency and biofilm formation between 

3.4 g/l and 35 g/l. Salinity influenced the formation of 

biofilms even at values below 5 g/l, the number of 

adherent bacteria in the biofilm formed at 11g/l 

salinity being well over the expected one. At 4g/l 

salinity there is a decrease in the number of cells from 

3.5∙10 6 CFU/cm 2 to 3.8∙10 4 CFU/cm 2 five days after 

immersion. 

Some experiments regarding the role of 

bacterial biofilm were accomplished by [35] in the 

mediation of invertebrate attachment and 

microfouling formation at different temperatures and 

salinity values of 20g/l-34g/l. There is bacterial 

increase from 12.8∙10 3 cel/mm -2 to 20g/l la 21.2∙10 3 

cel/mm -2 at 34 g/l. The experiments revealed no 

significant correlation between salinity and bacterial 

density in regards to biomass. 

Some experiments were accomplished in 

regards to the role of salinity (between 12g/l and 

80g/l) in the surface corrosion [37] achieves some 

experiments using stainless steel as substrate. They 

revealed the existence of a drop of cellular density 

with the increase of water salinity, noticing a 

corrosion maximum at 35 g/l between 1.7 ∙10 9 

CFU/cm 2 and 2.1∙10 CFU/cm 2 for the aerobe species 

152 

analyzed. The experimental data have increased 

values compared to those obtained by our 

experiments. 

The values of cellular density emphasize an 

increase correlated with the modification of salinity 

value as a whole, salinity increase from 5g/l to 10g/l 

and to 15g/l, the normal average value for seawater. 

These data are confirmed by the specialty literature as 

long as the increase is recorded between certain 

optimum salinity limits. 

The density values obtained when salinity was 

modified to 5g/l are lower than those for salinity from 

10g/l and 15g/l, which demonstrates that a possible 

supply of fresh water in the natural environment may 

influence the formation manner of the biofilms. 

Microcolonies form from the very first hours 

after the immersion of the hydrophile surfaces in the 

case of salinity variation as well. These data are 

confirmed by [24] in the specialized literature in the 

case of experiments for the formation of biofilms in 

static conditions. 


The environmental factor such as temperature 

and salinity seems to influence bacterial adherence. 

The formation of the initial layers of the 

biofilms and their temporal dynamics in “in vitro” 

conditions determines a progressive increase of 

cellular density and the formation of microcolonies 

from the first hour after immersion in liquid medium. 

The modification of temperature and salinity values 

determined a decrease of the total number of adherent 

cells, compared to the normal one on the hydrophile 

surface of glass, by mechanism(s) which are under 

investigation. 

5. References 

[1] ZOBELL C.E., 1943 – The effect of solid 

surfaces upon bacterial, J. Bacteriol., 46 (1): 39– 

56. 

[2] COSTERTON J.W., GEESEY G.G., CHENG 

K.J. 1978 – How Bacteria Stick, Scientific 

American; 238 (1): 86-95. 

[3] ZARNEA GH., 1994 – Tratat de Microbiologie 

generalã, Ecologia microrganismelor, Vol. 5, 

Ed.Academei Române, Bucureşti.


[4] LAZAR V. 2003- Aderenţa microbianã, 

Academiei Române, Bucureşti , 216 pp. 

[5] ELSE T. A., PANTLE C. R., AMY P. S., 2003- 

Boundaries for Biofilm Formation: Humidity and 

Temperature, App. .and Environ. Microbiology, 

69(8): 5006–5010. 

[6] CARLUCCI A. F. AND PRAMER D., 1959- 

Factors Affecting the Survival of Bacteria in Sea 

Water, Microbiological Process Report, 7: 388- 

392 

[7] DUFOUR PH., 1982 - Influence des conditions 

de milieu sur la biodéradation des matieres 

organiques dans une lagune tropicale 

Oceanologica Acta, 5 ( 3): 355-363. 

[8] ROSZAK D.B. AND WELL C., 1987 – Survival 

Strategies of Bacteria in the Natural 

Environement, Micro. Rev. 365-379. 

[9] COSTERTON J.W. 2007- The biofilm Primer, 

Springer, 200 pp. 

[10] PARSEK M. R. GREENBERG E.P., 2008 - 

Sociomicrobiology: the connections between 

quorum sensing and biofilms Trends in Microbioy , 

13 ( 1): 28-33. 

[11] ZOBELL C. E. AND CONN J. E., 1940 - 

Studies on the thermal sensitivity of marine 

bacteria, J. Bacteriol., 40 (2): 223–238. 

[12] MITTELMAN M.W.1998 - Structure and 

Functional Characteristics of Bacterial Biofilms, 

Dairy Sci., 81: 2760–2764. 

[13] COMPERE C., 1999 - Biofilms en mileu marin, 

Techniques Sciences Methodes, 11: 48-54. 

[14] WHITE-ZIEGLER C. A., SUZIN U., PEREZ N. 

M., BERNS A. L., MALHOWSKI A.J. AND 

YOUNG S., 2008- Low temperature (23ºC) 

increases expression of biofilm, cold-shock- and 

RpoS-dependent genes in Escherichia coli K-12, 

Microbiology, 154 : 148–166. 

[15] EDDLEMAN H., 1998 - Optimum Temperature 

for Growth of Bacteria, Indiana Biolab, 14045 

Huff St., Palmyra IN 47164 

[16] STANLEY S. 0. AND MORITA R. Y., 1968 - 

Salinity Effect on the Maximal Growth 

Temperature of Some Bacteria Isolated from 

Marine Environments, J. of Bacteriology, 95 (1) 

: 169-173. 

[17] HENRICI, A.T., 1933 -Studies of Freshwater 

Bacteria: I. A Direct Microscopic Technique. 

153 

[18] LEWANDOWSKI Z. AND STOODLEY P., 

1995 - Flow induced vibrations, drag force, and 

pressure drop in conduits covered with biofilm, 

International IAWQ Conference Workshop on 

Biofilm Structure, Growth, and Dynamics, 

Noordwijkerhout, The Netherlands. 

[19] POSCH T., FRANZOI J., PRADER 

M.,.SALCHER M., 2009 - New image analysis 

tool to study biomass andmorphotypes of three 

major bacterioplankton groups in an alpine lake, 

Aquatic Microbial Ecology, 54 : 113–126. 

[20] COSTERTON J.W, GEESEY G.G, CHENG 

K.J. - 1978 How Bacteria Stick, Scientific 

American, 238 (1): 86-95 

[21] http://www.BiofilmsONLINE.com, 2008: 

Techniques & Protocols, Henrici’s Microbial 

Capture Technique. 

[22] LAZAR V., HERLEA V., CERNAT R., 

BALOTESCU M., BULAI D., MORARU A., 

2004 - Microbiologie generala, Manual de 

lucrarii parctice, Universitatii Bucuresti, 320 pp. 

[23] HOVANEC T. A. AND DELONG E. F., 1996- 

Comparative Analysis of Nitrifying Bacteria 

Associated with Freshwater and Marine Aquaria, 

Appl. Environ. Microbiol, 62 (8) : 2888–2896. 

[24] MERRITT J. H., KADOURI D. E., AND. 

O’TOOLE G. A 2005- Growing and Analyzing 

Static Biofilms, Current Protocols in 

Microbiology, 1B.1.1-1B.1.17. 

[25] KUMAN A. AND PRASAD R., 2006 - Biofilms 

Jk. Science., 8 (1): 14 -17. 

[26] RUBIO C., 2002 - Comprehension des 

mecanismes d’adhesion des biofilms en milieu 

marin en vue de la conception de nouveaux 

motens de prevention, These de dcotorat, Paris,. 

216 pp. 

[27] SONAK S,BHOSLE N., 1995- A simple method 

to assess bacterial attachment to surfaces, 

Biofouling, 9(1): 31-38 

[28] HULEA A 1969 - Ghid pentru labortoarele de 

micologie şi bacteriologie. Ed. Agrosilvică, 

Bucureşti. 

[29] FRY, J.C., 1990 – Direct methods and biomass 

estimation. Meth. Microbiol, 22: 41-85. 

[30] COCIASU A., LAZAR L. AND VASILIU D. 

2008 –New Tendency in nutrient evolution from 

romanian costal waters, Cercetarii marine 

INCDM, 38: 7-23.


[31] ROGERS J., DOWSETT A. B., DENNIS P. J., 

LEE J. V., AND KEEVIL C. W., 1994 - 

Influence of Temperature and Plumbing 

Material Selection on Biofilm Formation and 

Growth of Legionella pneumophila in a Model 

Potable Water System Containing Complex 

Microbial Flora, Appl. Environ. Microbiol., 60 

(5): 1585-1592. 

[32] KELLEY J. I., TURNG B.F, WILLIAMS H. N., 

AND BAER M. L., 1997 - Effects of 

Temperature, Salinity, and Substrate on the 

Colonization of Surfaces In Situ by Aquatic 

Bdellovibrios, Appl. Environ. Microbiol, 63: 84- 

90. 

[33] DI BONAVENTURA G., STEPANOVIC S., 

PICCIANI C., POMPILIO A., PICCOLOMINI 

R., 2007- Effect of Enviromental Factors on 

Biofilm Formation by Clinical 

Stenotrophomonas maltophilia isolates, Folia 

Microbiol., 52 (1): 86-90. 

[34] TOREN L.A.., LANDAU L. KUSHMARO A., 

LOYA Y. AND ROSENBERG E., 1998 - 

Effect of Temperature on Adhesion of Vibrio 

Strain AK-1 to Oculina patagonica and on Coral 

Bleaching , Appl. Environ. Microbiol, 64 (4): 

1379–1384. 

[35] LAU S.C.K, THIYAGARAJAN V., CHEUNG 

S.C.K, QIAN P.Y., 2005 - Roles of bacterial 

community composition in biofilms as a 

mediator for larval settlement of three marine 

invertebrates, Aquat Microb. Ecol, 38: 41–51. 

[36] DI BONAVENTURA G., PICCOLOMINI R., 

PALUDI D., D’ORIO V., VERGARA A., 

CONTER M. AND IANIERI A., 2007- 

Influence of temperature on biofilm formation by 

Listeria monocytogenes on various food-contact 

surfaces: relationship with motility and cell 

surface hydrophobicity, J. Appl. Microbio., 104 : 

1552–1561. 

[37] FRANCA F.P., FERREIRA C.A., 

LUTTERBACH M.T.S., 2000- Effect of 

different salinities of a dynamic water system on 

biofilm formation, J. of Industrial Micro. & 

Bioteh., 25: 45-48. 

154

Density (10 

2 cel/mm 2 ) 

Density (10 

2 cel/mm 2 ) 


40 

35 

30 

25 

20 

15 

10 

5 

Early succesion of biofilms in containers over a period of 12 hours 

T 0=0 hours 

Slides Sea Water( Temp.6ºC) 

Slides Sea Water( Temp.18ºC) 

y = 2.3022x + 6.7253 

R 2 = 0.9354 

Table 1. The values of the seawater chemical parameters (liquid culture medium) 

Chemical parameters Sea water (zona litorala) Sea water 

(Aquarium) 

salinity 15.10 g/L 25.10 g/L 

pH 8.12 unit. 6.56 unit. 

P-PO4 0.74 µmoli/dm 3 63.80 µmoli/dm 3 

N-NO2 0.42 µmoli/dm 3 12.51 µmoli/dm 3 

N-NH4 1.13 µmoli/dm 3 5.46 µmoli/dm 3 

N-NO3 3.14 µmoli/dm 3 30.25 µmoli/dm 3 

Si-SiO4 21.16 µmoli/dm 3 0.24 µmoli/dm 3 

y = 1.9066x + 1.7912 

R 2 = 0.9668 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Time (hours) 

Density (10 2 cel/mm2 ) 

155 

66 

61 

56 

51 

46 

41 

36 


T 0=24 hours 

Slides Sea Water ( Temp.6ºC) 

Slides Sea Water (Temp.18ºC) 

y = 1.25x + 43.107 

R 2 = 0.9895 

y = 1.1429x + 35.714 

R 2 = 0.9922 

0 2 4 6 8 10 12 14 

Time (hours) 

Fig.1. The formation of a biofilm under the influence of temperature in containers with littoral seawater 

Ealy succesion of biofilms in containers over a period of 12 hours 

T 0=0 hours 

y = 2.6429x + 1.9121 

R 2 y = 2.5165x + 10.363 

R 

= 0.9883 

2 45 

40 

35 

Slides Aquarium (Temp.6ºC) 

Slides Aquarium ( Temp.18ºC) 

30 

25 

20 

= 0.8844 

15 

10 

5 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Time (hours) 


65 

60 

55 

50 

45 

40 


T 0=24 hours 



y = 1.125x + 47.839 

R 2 = 0.9883 

y = 1.2321x + 39.661 

R 2 = 0.9919 

0 2 4 6 8 10 12 14 

Time (hours) 

Fig.2. The formation of a biofilm under the influence of temperature in the containers with aquarium seawater


Density (10 

2 cel/mm 2 ) 

40 

35 

30 

25 

20 

15 

10 

5 

Ealty succesion of biofilms in containers over a peiod of 12 hours 

T 0=0 hours 

Slides Sea Water(Sal.10g/l) 

Slides Sea Water ( Sal.15g/l) 

y = 2.3022x + 6.7253 

R 2 = 0.9354 

y = 2.4341x - 0.2198 

R 2 = 0.9793 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Time(hours) 

156 


65 

60 

55 

50 

45 

40 

Ealry succesion of biofilms in containers over a period of 12 hours 

T 0= 24 hours 



y = 1.125x + 47.839 

R 2 = 0.9883 

y = 1.1607x + 39.446 

R 2 = 0.9816 

0 2 4 6 8 10 12 14 

Time (hours) 

Fig.3. The formation of a biofilm under the influence of salinity decrease (from 15g/l to 5 g/l) in the containers with 

littoral seawater 

Density (10 2cel/mm2 ) 

40 

35 

30 

25 

20 

15 

10 

5 


T 0= 0 hours 

Slides Sea Water ( Sal. 5g/l) 

Slides Sea Water (Sal.15g/l) 

y = 2.3022x + 6.7253 

R 2 = 0.9354 

y = 1.5385x - 0.2308 

R 2 = 0.9926 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Time(hours) 


69 

64 

59 

54 

49 

44 

39 

34 

29 

Early succesion of biofilm in containers over a period of 12 hours 

T 0=24 hours 

Slides Sea Water ( Sal.5g/l) 

Slides Sea Water (Sal.15g/l) 

y = 1.125x + 47.839 

R 2 = 0.9883 

y = 1.4286x + 28.714 

R 2 = 0.9627 

0 2 4 6 8 10 12 14 

Time (hours) 

Fig.4. Biofilm formation under the influence of salinity decrease (from 15g/l to 10g/l) in containers with littoral 

seawater


THE CLINICAL UTILITY OF ADITIONAL METHODS IN EFFUSIONS 

EVALUATION 

Ana Maria CRETU * , Mariana ASCHIE ** , Diana BADIU ** , Natalia ROSOIU *** 

*Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology, 

Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: cretu_anamaria@yahoo.com 

** Clinical Emergency Hospital of Constanta, Department of Pathology, Tomis Avenue, No. 145, 900591, 

Constanta 

***Ovidius University of Constanţa, Medicine Faculty,Mamaia Avenue, No. 124, Constanţa, 900527, Romania 

________________________________________________________________________________________ 

Abstract: Cells from reactive or hyperplasic mesothelium shed from body cavity surface, in various biological 

conditions, may present a wide range of deviation from normal cellular morphology, making it difficult, or even 

impossible, to distinguish them from malignant cells by mean of purely cytological criteria. This study was 

carried out with the aim to evaluate if macroscopic features and cytologic formula can be used as potential 

diagnostic tool for distinguishing between malignant cells from reactive mesothelial cells in peritoneal effusions. 

We have examined the peritoneal effusions collected from 81 available cases, with a histological diagnosis 

known, from routine morphologic features. The various macroscopic parameters that were registered by 

macroscopic analysis included the registration of color, transparency and fluidity of peritoneal effusions. 

Comparing the results, there wasn`t found any relationship between peritoneal fluid containing cancer cells and 

liquid color. Cell smear appearance had a various cells populations and the quantitative analysis of effusions was 

not enough useful in establishing the final diagnosis. 

Keywords: peritoneal effusions, macroscopy, cytology, malign, benign 

__________________________________________________________________________________________ 


The cytological diagnoses of serous effusions 

are usually made by routine cytomorphology with 

certainty, allowing treatment decisions. Various 

studies have shown a sensitivity of 57.3% and 

specificity of 89% by conventional cytology for the 

detection of malignant cells in effusion samples [1]. 

The conventional cytology rate for identification 

of neoplastic cells in effusions is about 60%. The rate 

of diagnostically equivocal effusions in routine 

cytology is dependent on the volume of effusion 

examined, type of preparation and staining, 

experience of the examiner, and application of 

ancillary methods [2]. Peritoneal effusions are a 

frequently encountered clinical manifestation of 

metastatic disease, with breast, ovarian, and lung 

carcinomas and malignant mesothelioma leading the 

list [3, 4]. 

Neoplastic cells that disseminate into cavities 

containing effusions are highly metastatic and possess 

a strong autonomous proliferative drive while 

concurrently being stimulatory of exudative 

effusions. The diagnosis of a malignant effusion 

signifies disease progression and is associated with a 

worse prognosis regardless of the tumor site of origin. 

Furthermore, cancer cells of different origins differ 

considerably in their biology and have unique 

phenotypic and genotypic characteristics [5]. 

Primary cytomorphologic criteria of malignancy 

include cellular aggregates, pleomorphism (variable 

cellular appearance), anisocytosis (variation in cell 

size), anisokaryosis (variation in nuclear size), 

multinucleation, prominent to irregular nucleoli, 

increased nuclear to cytoplasmic ratio, monomorphic 

cellular appearance, and increased mitotic figures. 

Hyperplastic mesothelial cells also may exhibit 

anisocytosis, anisokaryosis, increased nuclear to 

cytoplasmic ratio, binucleate and multinucleate, and 

scattered mitoses. Any situation that results in fluid 

accumulation within the body cavities can induce 

mesothelial cell hyperplasia and exfoliation with an 


The clinical utility of aditional methodes... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) 

abnormal cellular morphology [2]. Therefore, the 

differentiation between mesothelial cell hyperplasia 

and mesothelioma may be difficult or impossible. 

The first report of an intraoperative examination 

of peritoneal cytology to detect subclinical metastases 

was presented in 1971. Patients with normal 

peritoneal cytological specimens had better survival 

rates than patients with abnormal findings, but only 

one abnormal cytologic specimen was found in early 

stage disease. [6]. Factors such as in patient versus 

outpatient management and associated procedural 

discomfort are important in the decision making 

process, and the patient should participate in these 

subjective considerations [7]. 

In addition, the etiology of the primary 

complaint is frequently multifactorial. However, 

malignant effusions recur, and therefore repeated 

paracentesis, especially if the fluid rapidly 

reaccumulates, is usually not a good long-term 

solution unless the patient’s overall prognosis and 

current condition prohibits a more invasive option. 

It is difficult to compare results and determine 

the true efficacy of different techniques and agents 

because endpoints and response criteria as well as the 

extent and method of follow-up vary. Therefore 

various techniques should be used to increase the 

diagnostic accuracy of malignancy in serous 

effusions. 


This study was based on evaluation of 81 

available cases, with a histological diagnosis known, 

carried out in Emergency Clinical Hospital of 

Constanta – Pathological Anatomy Department 

(SCJUC ) from octobre 2007 to January 2010. 

Follow-up data were obtained from the Tumor 

Registry at SCJUC. Clinical charts of all the patients 

whose peritoneal fluid samples were sent for 

cytological examination during the study period were 

retrieved for relevant information. 

The fluid for cytological analysis was collected 

during laparotomy from the abdominal cavity. If no 

fluid was present, the peritoneal cavity was lavaged 

with saline solution, and the fluid was then collected 

for analysis. 

Giemsa stained and Papanicolaou stained slides 

were prepared from sediment obtained by 

158 

centrifuging the peritoneal liquid samples at 1500 

rpm for 5 minutes, using Shandon Cytospin 

preparations. After the centrifugation, the stained is 

fixed using alcohol (95% ethyl alcohol) as the 

fixative. Effusion cytology was studied from 40 

peritoneal effusions associated with at least one 

malignancy and 41 effusions collected from pacients 

with hepatic cirrhosis. 

We have examined the peritoneal effusions 

from routine macroscopic and cytologic features. 

Determination (the qualitative method) of 

cellular density, specific weight and protein content 

from peritoneal fluid was performed by the Riwalta 

reactions [61]. Riwalta reaction is the reaction 

performed for differential diagnosis of exudates from 

transudates, based on precipitation of fibrin 

(insoluble protein, the main component of blood clot, 

a result of thrombin action on fibrinogen in plasma 

soluble, synthesized by the liver) meeting usual in 

inflammatory exudates (transudates usual, do not 

contain this fibrin) [10]. The reaction is positive 

when dripping the liquid examined in the mixture is 

obtaining an opalescent, as a cloud. For obtaining the 

liquid peritoneal cytology formula, 100 cellular 

elements were measured from each smear cellular, 

thus directly establishing a percentage value. 


From all 81 cases who developed peritoneal 

fluid, 41 were benign cases (associated with liver 

cirrhosis), 4 cases were associated with hepatic 

carcinoma, 4 cases with lung carcinoma, 18 cases 

with ovarian carcinoma, 4 cases with breast 

carcinoma, 9 cases with gastrointestinal carcinoma 

tract and 2 cases with peritoneal mesothelioma (Table 

1). The studied cases were divided into two groups: 

the 41 benign cases were included in lot I and 40 

cases of peritoneal fluid associated with cancer were 

included in lot II. 

Table 1. Peritoneal fluid distribution according to 

primary disease and the number of cases 

Lots Primary disease No of 

cases 

Lot I Hepatic cirrhosis 41 

(no=41)

Lot II 

(no=40) 

Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) 

Hepatic cancer 4 

Ovarian cancer 18 

Gastrointestinal cancer 9 

Breast cancer 4 

Pulmonary carcinoma 3 

Peritoneal mesothelioma 2 

In terms of etiology, the highest number of cases 

groupt in lot II were shown to have ovarian origin, 

represented by ovarian carcinoma (n = 18) (45%), 

followed by cases associated with gastrointestinal 

carcinoma (n = 9) (22.5%), liver and breast 

carcinoma (n = 4) (10%), carcinoma lung (n = 3) 

(7.5%) and malignant mesothelioma (n = 2) (5%) 

(Fig.1). 

The type of neoplasia associated with most 

cases with peritoneal effusions accumulation proved 

to be represented by ovarian carcinoma. 

Fig. 1. Percentage distribution of cases according to 

the origin of cancer associated 

Most patients in Group II (32.83%) were within 

the range of ages 61-70 years (40%), followed by the 

51-60 range (35%). Cases registered with ovarian 

carcinoma were included in most (61.11%) in the 51- 

60 age range, those registered with gastrointestinal 

carcinoma and the liver were contained mainly in the 

41-50 age range, breast cancers, malignant 

mesothelioma and lung were within the range 61-70. 

(Fig. 2). 

159 

Fig. 2. The peritoneal fluid on different intervals of 

age (HC hepatic cancer, OC ovarian cancer, GIC 

gastrointestinal cancer, BC breast cancer, PC lung 

cancer, MM malignant mesothelioma) 

According to cancer staging [8], 

- stage I: generally include small tumors without 

invasion and who are perfectly curable in most cases 

the prognosis favorable 

- stage II and III includes tumors with local 

invasion of surrounding tissues and lymph nodes, 

therapeutic approach and prognosis are different 

depending on the time cells and organ of origin, 

- stage IV: at this stage are in general inoperable 

tumors, metastasis and recurrence and a reserved 

prognostic survival, only 7 cases (17.5%) (2 / 2, and 

peritoneal mesothelioma 100% 5 / 18, 27.77% 

ovarian carcinoma) were rated as stage III, the rest 

(82.5%) fits into state IV, which shows that, as the 

stage progresses neoplasia, this is more prevalent 

peritoneal fluid (Fig.3). 

Fig. 3. Percentage of cases according 

to stage neoplasia


From all patients with histopathologic and 

clinical data that indicate malignancy, a number of 5 

patients (2 / 2, 100% associated with malignant 

mesothelioma, 1/4, 25% associated with hepatic 

carcinoma, ¼, 25% associated with breast carcinoma, 

1/3, 33.33% associated with lung carcinoma) were 

deceased before drawing to the final study (a period 

of approximately three years from the accumulation 

of fluid in the peritoneal cavity), in all cases, the 

peritoneal fluid cytology recorded the presence of 

malignant cells (Fig. 4). 

Fig. 4. Percentage of prognostic survival of patients 

included in the study (OC- ovarian cancer, GIC - 

gastrointestinal cancer, HC - hepatic cancer, BC – 

breast cancer, PC - pulmonary cancer, MM - 

malignant mesothelioma) 

Thus, the percentage of peritoneal fluid 

accumulation in the abdominal cavity is directly 

proportional to the tumor stage and also with the 

diagnosis of malignant peritoneal effusions, meaning 

that the progression of cancer is associated with an 

unfavorable prognosis. None of the patients with 

ovarian or gastric carcinoma were associated with an 

unfavorable prognosis, which indicates that this 

patients are available for a longer treatment. 

Neoplasia stage, histological grade of neoplasia, 

positive cytology of peritoneal fluid and patients age 

(61-70 years) were correlated statistically with the 

prognosis. 

The first step in the analysis of peritoneal fluid 

was represented by analysis of the macroscopic point 

of view of biological material (peritoneal effusions). 

160 

Thus, it was recorded: the extracted amount, the 

product color, transparency and its consistency. 

After macroscopic analysis, the most liquids 

from the group I was found to shown yellow color 

(from very light yellow to orange - yellow), and most 

fluid were transparent. In stead, the peritoneal 

effusions from group II had a variable macroscopic 

appearance: a number of 29/40 (72.5%) were intense 

yellow colored and transparents, many of them 

(17/29, 42.5% of all liquids associated with a 

carcinoma) had tissue fragments occupying 

approximately 25-50% from all quantity effusions, 

suspended in liquid; 5 (12.5%) showed a yelloworange 

fluid and opaque, and a total of six (15%) 

were hemorrhagic (deep red), fluid and opaque (Fig. 

5). Of these, 19 / 40 (47.5%) had fragments of tissue 

suspended in peritoneal effusions. These fragments 

were then included in paraffin, stained and examined 

microscopically. 

We can say that the macroscopic analysis of 

peritoneal fluid, associated with cases of cancer are 

different from those associated with cases of cirrhosis 

only by this tissue fragments founded suspended in 

the effusions, with a capacity of discrimination of 

47.5%. 

Fig. 5. Peritoneal fluid: yellow (a) and hemorrhagic 

The registration of the important differences 

existing between macroscopic peritoneal fluids is 

essential, representing the first way in effusions 

discrimination (in transudates and exudates), so that, 

the material subjected can provide useful information 

for further evaluation of the cells from cytological 

smears. It is known that bleeding effusions (deep red) 

are often caused by a cancer and that these liquids 

often contain cancer cells [9].

Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010) 

However, comparing the results, after 

performing peritoneal fluid cytology, with those 

obtained by macroscopic evaluation, of the 40 

effusions associated with at least one malignancy, 

only 6 (15%) were founded to be red colored 

(hemorrhagic), emphasizing that it does not exist any 

relationship between peritoneal fluid containing 

cancer cells and fluid color. 

After conducting the Riwalta reactions [10], the 

81 peritoneal effusions were classified in: 29 

(35.80%) transudates peritoneal fluid (with low cell 

density and low protein content, which is usually 

accumulated in benign conditions) and 43 (53.08%) 

exudate (effusions with high cell density and high 

protein content, which is accumulated most in 

malignant conditions), and 9 (11.11%) mixed, 

intermediate peritoneal effusions. Thus, peritoneal 

fluids were classified into three groups: group I 

(transudates), group II (intermediate, mixed) and 

group III (exudates) (Table 2). 

Table 2. Distribution of cases after Riwalta reaction 

Lots Primary 

cancer 

Lot I 

(n=41) 

Lot II 

(n=40) 

Transudates 

(N=29) 

Mixed 

(N=9) 

Exudates 

(N=43) 

CB 21 3 17 

CH (4) 1 1 2 

CO (18) 3 0 15 

CGI (9) 1 2 6 

CM (4) 2 2 0 

CP (3) 1 1 1 

MP (2) 0 0 2 

Since only 26/40, 65% of peritoneal effusions 

associated with different type of cancer resulted to 

have characters of exudates, and only 21/41, 51.21% 

of effusions associated with liver cirrhosis were 

shown to be transudates, it follows that, by 

conducting the Riwalta reaction, it can determine the 

benign or malignant nature of effusions in a 

proportion of 58.10% (Fig.6). 

Cell smear appearance had a various cells 

populations and the quantitative analysis of effusions 

was not enough useful in establishing the final 

diagnosis. There were observed 7 cell types present 

in variable number. Proliferative capacity of tumor 

cells - tumor aggressiveness - is an important 

161 

element in cytology grading of malignancy, and was 

quantified by mitosis counting. 

Fig 6. The benign or malignant nature of effusions 

after conducting the Riwalta reaction 

In smears classified as benign, isolated cells 

represented 90% of total cells, cell groups recovered 

to a rate of 10%. 5% were represented by free 

nucleus or cells with damaged cytoplasm. 

Mesothelial cells (33%) (33 cells of 100 

elements) and lymphocytes (30%) were the majority 

cell type in the group of benign peritoneal effusions, 

followed by macrophages (17%), polymorphonuclear 

leukocytes (PMN) (9%) and erythrocytes (7%). 

Average of total number of mitosis founded in 

studied smears was 3mitosis/smears (Table 3). 

Cellular composition of effusions founded to 

be suspicious for malignancy was similar with the one 

of benign peritoneal effusions: mesothelial cells 

(28%) (28 cells of 100 items) and lymphocytes (26%) 

were the majority cell type, followed by neutrophils 

(13%), atypical cells, suspicious for malignancy 

(9%), erythrocytes (9%) and macrophages (7%). 

Average of total number of mitosis founded in 

studied smears was 7 mitosis/smears (Table 3). 

In the group of patients with malignant cancer, 

mesothelial cells represented 29% and erythrocytes 

21%, followed by lymphocytes (18%), 

polymorphonuclear leukocytes (16%), macrophages 

(4%) and malignant cells (4%), average of total 

number of mitosis was 9 mitoses / cell smear (Figure 

7).


Tabel 3. The percentage of cellular elements (%) 

BE* AE* ME* 

Mesothelial 

cells 

33 28 29 

Atipical cells 0 9 0 

Malignant cells 0 0 3 

erythrocytes, 7 9 21 

lymphocytes 30 26 18 

PMN 9 13 16 

macrophages 17 7 4 

mitosis / cell 

smear 

2 7 9 

Average 12,25 12,375 12,5 

standard 

Deviation 

13,15566 9,738546 10,12776 

p(t


SPATIO-TEMPORAL DYNAMICS OF PHYTOPLANKTON COMPOSITION AND 

ABUNDANCE FROM THE ROMANIAN BLACK SEA COAST 

Laura BOICENCO 

National Institute for Marine Research and Development „Grigore Antipa” 

300, Mamaia Bd., Constanta, 900581, Romania, e-mail: boicenco@alpha.rmri.ro 

__________________________________________________________________________________________ 

Abstract: Based on more than 2,000 samples collected during 1996-2007, the paper deals with the taxonomic 

and ecological composition, spatio-temporal development of phytoplankton blooms from waters of up to 50 m 

depths laying on the Romanian Black Sea. The author identified 396 species, varieties and forms, and assessed a 

density mean varying among 417 and 3,376∙10 3 cells∙l -1 . Bacillariophyta phylum, with a number of 157 taxa and 

a density mean of minimum 186.4 (in 2000) and maximum 2,311.9∙10 3 cells∙l -1 (in 1997), was the most numerous 

(39.6% of the total); Dinoflagellata was the second dominant group, represented in the communities with 85 taxa 

(21.5%); density means ranged from 9.2 (in 2003) and 225.6∙10 3 cells∙l -1 (in 1997). Groups Chlorophyta and 

Cyanobacteria represented only 19.4 and 12.9%, respectively, from the total number of species. Species showing 

huge developments in the reference period were: the diatoms Skeletonema costatum, Cerataulina pelagica, 

Nitzschia delicatissima, Chaetoceros socialis, Cyclotella caspia and dinoflagellates Prorocentrum minimum, 

Heterocapsa triquetra and Scrippsiella trochoidea. 

Keywords: taxonomic composition, ecological composition, phytoplankton blooms 

__________________________________________________________________________________________ 


The early 1990s seemed to be a new 

beginning for the Black Sea ecosystem. After the 

“Mnemiopsis era” describing the 8 th decade, 

superimposed on the 20 years-long “eutrophication 

era” started in the 1970s, signs of improvement of 

its ecological state occurred, evidenced by a 

reduction of the Danube river nutrient input, a 

decrease in the frequency of hypoxia conditions, an 

increase in fodder zooplankton biomass, and a 

drop in M. leidyi’s abundance. The recovery of the 

ecosystem was attributed partly to the collapsing 

economy and agricultural production, and to some 

protective measures taken to control anthropogenic 

pollution in all the coastal countries. 

Due to their short life cycles and quick 

response to changes in their environment, the 

phytoplankton was sensitive to these new shifts, 

displaying a tendency to “normal” status before 

eutrophication: decreased amplitude and frequency 

of blooms, and a qualitative and quantitative 

structure similar to the period 1960-1970 rather 

than 1980-1990. 

So, between 1991 and 1996, only six maximum 

densities, higher than 10 6 cells·l -1 , were registered, 

compared to 13 in the1980s; among them, only two 

species produced ample bloom events: Prorocentrum 

minimum (53.1 and 93.7·10 6 cells·l -1 in the summer of 

1991 and 1995) and Microcystis pulverea (60.0·10 6 

cells·l -1 in the spring of 1991) [1]. 

The range of algal groups was different from that 

of 1970s and 1980s, but quite similar to that of 1960- 

1970 with a reduction of non-diatom bloom amplitude 

and increase of numerical density and especially 

biomass of diatoms. The reduction of non-diatoms 

coincided with decrease of nutrient stocks, especially 

of phosphates, which reached concentrations of 2.55 

µM·l -1 in 1991-1996, 2.8 times lower than in 1986- 

1990 [1]. 

During 1995-1996, the Black Sea ecosystem 

showed abrupt shifts in all trophic levels, from primary 

producers to apex predators. This arose as a 

manifestation of concurrent changes in its physical 

climate induced by intensive warming of surface 

waters, as well as abrupt increases in the mean sea 

level and annual mean fresh water flux [2]. 


Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169 

The aim of the present paper is to evaluate 

the spatio-temporal dynamics of the main 

taxonomic groups, and also the main bloomforming 

species during 1996-2007. 


Biological material was collected during 

seasonal surveys carried out within the scientific 

NIMRD’s programs, on board RV/STEAUA DE 

MARE, in the Romanian coastal waters laying 

between 43 0 50’-45 0 05’N and 28 0 50’–30 0 00’E (Fig. 

1). 2,018 quantitative samples were collected from 

472 stations covering the whole Romanian littoral 

at standard depths (0, 10, 20, 30, 40, 50 and 60m), 

from the following profiles: Sulina, Mila 9, Sf. 

Gheorghe, Zaton, Portiţa, Chituc, Constanta, 

Mangalia. 

Fig.1. Sampling network 

The sampling used a NISKIN bottle; the water 

is transferred in 500ml bottles and preserved with 

formaldehyde 4%. In laboratory, the samples were 

processed using the MOROZOVA-VODIANITSKAIA 

and BODEANU’s methods [3, 4]. After two weeks of 

sedimentation, the supernatant liquid is siphoned 

off up to about 100ml. The sample is put in a small 

jar for another 10 days sedimentation. Before 

164 

microscopic processing, the sample is again siphoned 

off up to 10ml and stirring. 0.1ml of sample is 

examined under a ZEISS inverted microscope; the cells 

are counted and identified at species or genus and the 

numerical density is obtained relating the number of 

cells to a volume of 1 litre. 

Table 1 presents the environmental background 

(inorganic nutrients) of the annual phytoplankton 

developments. Phosphates showed a sharp decrease 

after 1997 down to a level similar to that before 

eutrophication. The inorganic total nitrogen 

concentrations have steadily depleted ever since the 

1980s down to a minimum 9.48µM in 1985 followed 

by a relative long period, when these nutrients 

presented non-uniform oscillations. Among 1995 and 

2005, the levels of total inorganic nitrogen 

homogenously decreased, the maximum limit being 

situated between 10 and 15µM. In recent years their 

concentrations have began to increase to more than 

20µM, a value similar to that from 1980. 

With the exception of Si/N ratio, the molar 

ratios are still far by from the normal values, 

indicating that trophic anions do still not have optimal 

values for the normal development of marine 

phytoplankton, although they show a decreasing 

tendency. During the last period, the N/P ratio 

increased, due to an excessive decrease of phosphates 

and slight increase of inorganic nitrogen [5]. 

Table 1. Multiannual mean of surface nutrient 

concentrations in coastal waters off Constanta 

Period 1983-‘90 1991-‘00 2001-‘05 

N-NO3 (µM) 6.90 5.90 7.98 

N-NH4 (µM) 5.11 7.06 6.12 

P-PO4 (µM) 6.54 1.86 0.49 

SiO4 (µM) 11.0 12.6 13.7 


During 1996-2007, 396 microalgae species, 

varieties and forms belonging to seven phyla were 

identified in the Romanian Black Sea waters (Fig. 1), 

the minimum number of 140 being found in 1996 and 

the maximum one in 2004. The most important group 

is Bacillariophyta, with 157 species, representing 

39.6% of the total; the second place is occupied by 

dinoflagellates, with 85 species (21.5%), followed by

Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169 

chlorophytes – 77 species (19.4%) and 

cyanobacteries – 55 species (12.9%); the rest of 

phyla (Chrysophyta, Euglenophyta, Cryptophyta), 

with 12, 8 and 6 species, respectively, constitute 

together only 6.6% of the total (Fig. 2). 

Fig. 2. Taxonomic composition. 

Ecologically, the phytoplankton represents a 

combination of autochthonous species, comprising 

euryhaline marine and brackish water forms (218), 

and alochthonous forms, comprising fresh-brackish 

and fresh water forms (178), reflecting the mixed 

marine water masses and riverine fresh waters 

which characterise the hydrologic regime of the 

Romanian sector. 

Table 2. Structure by ecologic groups. 

Phylum MarineFreshbrackish brackish 

Bacillariophyta 112 45 

Dinoflagellata 83 2 

Chlorophyta 0 77 

Cyanobacteria 9 42 

Chrysophyta 8 4 

Euglenophyta 2 6 

Cryptophyta 4 2 

Total 218 178 

Diatoms 

During 1996-2007 the averaged data for the 

whole Romanian littoral waters suggest that the 

communities are dominated by diatoms, both in 

terms of numeric density and biomass. The 

multiannual mean of 833.5·10 3 cells·l -1 is 12.2 

times higher than dinoflagellates (68.5·10 3 

cells·l -1 ). The highest diatoms mean density - 

2,311.9·10 3 cells·l -1 , achieved in 1997 was 10.2 

times higher than that recorded for dinoflagellates 

165 

in the same year. The diatoms produced their highest 

mean densities during summer; in the summer of 1997, 

they exceeded 6 million cells per litre. When we were 

able to collect samples in winter, e.g. in 1999, we 

found out that many diatoms – such as S. costatum, C. 

caspia, Ch. socialis, N. tenuirostris began to vegetate 

in winter. So, we detected communities very well 

constituted, higher than 10 6 cells∙l -1 . In five out of 12 

springs investigated (1999, 2003, 2004, 2006 and 

2007), the diatom populations were most abundant; 

they progressively decreased toward summer and 

autumn. 

Table 3 shows the diatom species with the 

highest densities in the Romanian Black Sea waters, 

between 1991 and 2007. 

Table 3. The highest densities of diatoms 

(10 6 cells∙l -1 ) 

1996- 2001- 

Species 

2000 2007 

Cyclotella caspia 10.5 78.6 

Skeletonema costatum 24.4 37.3 

Nitzschia tenuirostris 1.8 15.5 

Cerataulina pelagica 8.2 10.0 

Chaetoceros socialis 22.2 7.5 

Skeletonema subsalsum 4.4 3.9 

N. delicatissima 0.6 2.5 

Small-sized diatom, Skeletonema costatum is an 

omnipresent species in the communities identified not 

only at the Romanian littoral but in whole Pontic 

basin, producing most of the bloom events. Its 

maximum level was registered in July 2002, off 

Constanta (37.3∙10 6 cells∙l -1 ). Its second outburst 

occurred in the shallow waters of Mamaia Bay (where 

the sampling is carried out almost weekly); starting in 

the second half of March 1998, the phytoplankton 

communities were more and more abundant, attaining 

the value of 24.3∙10 6 cells·l -1 on March, 31. We have 

to remark that the communities from Mamaia Bay 

were almost monospecific, being constituted up to 

99.8% only by Skeletonema. 

Eurythermal and euryhaline species, 

Skeletonema vegetates abundant starting from winter, 

not only in Mamaia Bay, where usually attain over 

5∙10 6 cells∙l -1 , in January-March, but also in deeper 

waters off Constanta, where its concentrations reached


3.6∙10 6 cells∙l -1 in January 1999. In the spring of 

2006, again in Mamaia Bay, S. costatum produced 

two other bloom events: 15∙10 6 cells∙l -1 (April, 25) 

and 11∙10 6 cells∙l -1 (May, 4) (Fig. 3), when the 

temperatures oscillated from 9.8 to 13.3 0 C and the 

salinity decreased gradually from 16.53 to 12.64 

and 9.04 PSU. 

In waters under the direct influence of the 

Danube, Skeletonema often produced densities 

ranged from 1.4 to 6.1∙10 6 cells·l -1 , both in spring 

and summer. But in September 1999, its 

populations were even richer at all the stations of 

the profile: 6.0 (Sulina), 8.1 (Mila 9), 7.7 

(Sf.Gheorghe) and 6.3 ∙10 6 cells·l -1 (Portita). 

Fig. 3. Long-term evolution of S. costatum blooms. 

However, the last blooms produced by 

Skeletonema are much lower than those recorded 

in the period of maximum eutrophication. A good 

indicator of hypereutrophic waters, S.costatum 

showed overwhelming populations after 1970; for 

instance, between 1983 and 1986, S. costatum 

bloomed up to its highest value of 141.4∙10 6 cells∙l -1 

in April 1983. 

But, the most significant bloom event 

registered during the whole study period was 

generated by another diatom, Cyclotella caspia in 

the shallow waters of Mamaia. On May 3, 2001, it 

reached the value of 78.6∙10 6 cells·l -1 , which is 3.2 

times higher than S. costatum’s peak; the event was 

amplified by the abundant population of 

Skeletonema, raising the total density up to 

84.82∙10 6 cells∙l -1 . 

Cyclotella gave another two important 

outbursts, but they were 7.6 and 4.0 times lower 

than the preceding one: in June 1999 (10.4∙10 6 

cells∙l -1 ) at Mamaia, and June 2005 (19.7∙10 6 

cells∙l -1 ) at Constanta (Fig. 4). Rich populations 

were found also in the northern sector, but never as 

high as those found in the southern area; the richest 

166 

one was identified in waters from Sf.Gheorghe site 

(6.4∙10 6 cells·l -1 ). Anyway, these highest values are far 

from the exceptional development recorded by 

Cyclotella in 1981 (300∙10 6 cells∙l -1 ) [6]. 

Fig. 4. Long-term evolution of C. caspia blooms. 

Chaetoceros socialis is the third diatom with 

frequent occurrence and densities higher than 100∙10 3 

cells·l -1 , but only two blooms were higher than 10∙10 6 

cells·l -1 : in June 1997, in front of the Danube Delta 

(Mila 9) (15.9∙10 6 cells·l -1 ), and in May 2000, at 

Mamaia (22.2∙10 6 cells∙l -1 ) (Fig. 5). 

Ch. socialis is a new entry the list of bloomforming 

species. During the period 1971-1990, only 

Ch. similis f. solitarius developed large 

concentrations: 13.2∙10 6 cells∙l -1 (between 1970 and 

1980) and 21.5∙10 6 cells∙l -1 in May 1988. 

Fig. 5. Long-term evolution of Ch. socialis blooms. 

However, in 1956, 1957 and 1961, SKOLKA cited 

Ch. socialis among the species producing some 

abundances higher than 10 6 cells·l -1 (its peak of 

2.6∙10 6 cells∙l -1 was attained in June 1957); generally it 

accompanied other bloom-forming species such as 

S. costatum [7]. 

During the study period, another diatom group, 

including Cerataulina pelagica, Nitzschia 

delicatissima and N. tenuirostris, periodically 

contributed to increase the total phytoplankton 

abundances, and C. pelagica had a density range from 

3 to 10 million cells per liter. But only two of


Nitzschia’ species (N. tenuirostris and N. 

delicatissima) had occurred and had low densities. 

Apart from the N. tenuirostris’ single bloom 

produced in July 2006 in Mamaia Bay (15.5∙10 6 

cells∙l -1 ), the two species had densities higher than 

10 6 cells∙l -1 only in five and six years, respectively. 

N. tenuirostris started to vegetate intensely after 

1981, and reached its amplest bloom in the summer 

of 1989 - 74.8·10 6 cells·l -1 [6]. 

Dinoflagellates 

With a long-term mean of 68.5∙10 3 cells∙l -1 , 

the dinoflagellates comprised small percentages of 

the total phytoplankton, with a maximum of 17% 

in 2007; the highest mean density was almost 

225.6·10 3 cells·l -1 in 1997. However, during two 

springs (1998 and 2007) the populations of 

dinoflagellates were denser, with a density mean of 

455.9·10 3 cells·l -1 . Between 1996 and 2007 a few 

species had concentrations higher than 10 millions 

cells per liter (Table 4) in different areas and years. 

Table 4. The highest densities produced by 

dinoflagellates (10 6 cells∙l -1 ) 

1996- 2001- 

Species 

2000 2007 

Scrippsiella trochoidea 0.3 25.3 

Heterocapsa triquetra 13.6 16.0 

Gymnodinium cf. aureolum - 10.7 

Prorocentrum minimum 10.5 9.0 

Mass growth of the Prorocentrum minimum, 

causing the water to turn red, was recorded for the 

first time in the summer of 1974 along the 

Romanian littoral; the phenomenon was repeated in 

summers 1975 and 1976. Prorocentrum was the 

first dinoflagellate species reacting to the sudden 

decrease in salinity (monthly average reached 13 

PSU, at Constanta) and huge increase in the 

concentrations of phosphates and nitrates (18 and 

11 times respectively higher than the period 1959- 

1960). Presence of such extraordinary blooms had 

never been noticed before: 181.5 (1974), 78.7 

(1975) and 111.6∙10 6 cells∙l -1 (1976), in the 

southern coastal waters, from Navodari to 

Mangalia [8]. During the following decades, when 

167 

eutrophication got stronger and stronger, up to a 

climax from 1981-1990, the species attained even 

more prodigious proliferations, up to the value of 

807.6∙10 6 cells∙l -1 in July 1987. In fact, no other 

species would ever attain such densities as the 

Prorocentrum between 1971 and 1990. In the 

following years, the amplitude of Prorocentrum’s 

blooms decreased, but in July 1995 it reached a 

density of 93.7∙10 6 cells∙l -1 (Fig. 5), 8.6 times lower 

than its overwhelming density in July 1987 [1]. 

Fig. 5. Long-term evolution of P.minimum blooms. 

Beside the diatom Skeletonema, P.minimum is 

the second most common species in the whole Pontic 

basin giving some of the highest blooms, especially in 

the NW sector. In our study period, P. minimum 

continued to have massive developments, but much 

lower than the previous ones: in June 1999 –10.4∙10 6 

cells∙l -1 and July 2001 – 8.93∙10 6 cells∙l -1 , both of them 

in Mamaia Bay. Here, up to 2001, during warm 

months, the species’ populations frequently exceeded 

1 million cells per litre; then, the densities were lower 

and lower, sometimes disappearing from samples. 

Three other dinoflagellates reached 

concentrations higher than 10∙10 6 cells∙l -1 , namely 

Heterocapsa triquetra, Scrippsiella trochoidea and 

Gymnodinium cf. aureolum (Table 2). After 

developments, reaching a few or ten thousands cells 

per litre in the 1970s, H. triquetra and S. trochoidea 

came to the list of the bloom-forming species, the first 

with a value of 97.6 ∙10 6 cells∙l -1 in the period 1971- 

1980, and the second one with a value of 25.8 ∙10 6 

cells∙l -1 in the period 1981-1990. After a period (1991- 

1996) of insignificant concentrations (highest value of 

1.9 ∙10 6 cells∙l -1 ) [1], Heterocapsa again reached high 

concentrations: 13.6∙10 6 cells∙l -1 , in May 1998 (at Mila 

9) and 10.3∙10 6 cells∙l -1 , in April 2000 (in Mamaia 

Bay) (Fig. 6). All along Romanian littoral, but 

especially in the northern sector, Heterocapsa 

produced substantial densities, ranging from 2.0 to


5.0∙10 6 cells∙l -1 . S. trochoidea and Gymnodinium 

cf. aureolum had only one single large bloom 25.2 

(August 2001) and 10.1∙10 6 cells∙l -1 (April 2007 in 

Mamaia Bay), respectively. 

Fig. 6. Long-term evolution of H. triquetra 

blooms. 

Other Groups 

Representatives of other groups did not 

achieve significant densities, only sporadically did 

some species dominate the communities, and 

cyanobacteria were the most numerous comparing 

with chlorophytes and chrysophytes. The species 

Merismopedia, Microcystis, Gloeocapsa, 

Oscilatoria and Aphanizomenon were the 

commonest and most frequent cyanobacteries 

(Table 4). 

Table 4. The highest densities produced by other 

groups (10 6 cells∙l -1 ) 

Species 

1996- 

2000 

2001- 

2007 

CYANOBACTERIA 

Microcystis orae 272.0 

Microcystis pulverea 1.0 26.7 

M. aeruginosa 1.5 15.0 

Phormidium sp. 27 1.1 

CHRYSOPHYTA 

Emiliania huxleyi 1.3 1.1 

EUGLENOPHYTA 

Eutreptia lanowii 2.4 7.4 

Three species of Microcystis genus (M. 

pulverea, M. aeruginosa and M. orae) produced 

maximum densities between 12.8 and 271.9∙10 6 

cells∙l -1 , especially during summer of 2001-2003. 

The intense development of these three small-sized 

168 

species of cyanophytes took place under increased 

values of temperature, simultaneously with decreased 

values of salinity and concomitant with optimal 

concentrations of nutrients [8]. The euglenophyte 

Eutreptia lanowii had a constant frequency of 

occurrence throughout the analyzed period, with 

maximum developments in June 2007 of 7.4∙10 6 

cells∙l -1 , in Mamaia Bay, and 2.8∙10 6 cells∙l -1 July 2002 

off Constanta. 


Despite of the mitigation in the pressure exerted 

by anthropogenic eutrophication (i.e. depletion of the 

inorganic nutrient concentrations down pre 1970 

values) and Mnemiopsis’grazing, the signs of the 

ecosystem rehabilitation identified at the 

phytoplankton level occurred after 1990, seem to be 

very fragile and labile. That means if the necessary 

conditions (sudden salinity reduction, sudden increase 

in water temperature, high concentrations of specific 

biogenic compounds) are fulfilled, many of 

phytoplankters can produce ample blooms. 

Some of the species producing frequent and 

overwhelming blooms in the previous decades, carried 

on generating significant blooms also in our study 

period (i.e. S. costatum, P. minimum, C. caspia etc). 

Other species have newly entered the list of bloomforming 

species, especially small-sized cyanophyte – 

M. pulverea (occurred during the period 1991-1996), 

M. orae, M. aeruginosa, Synecocystis sp., Gloeocapsa 

crepidinium, but also some large-sized diatoms 

Navicula sp., Amphora sp., Tabellaria sp. (after 

1996); all of them are alochthonous fresh-brackish 

species, introduced into the sea mainly by the Danube 

River. M. orae gave a density of 272∙10 6 cells∙l -1 in the 

summer of 2000, the highest density occuring after 

1990. 

Many times in the past, some of the bloom 

events, especially these of huge concentrations, were 

followed by fish and invertebrate mass mortalities. We 

used to consider that the species blooming at the 

Romanian littoral were dangerous only due to the 

negative impact produced as consequence of oxygen 

depletion, reaching the threshold for lethal limits for 

invertebrates and fish. Such case took place in 1999, 

after a relatively high (10.4∙10 6 cells∙l -1 ) but longlasting 

bloom (June-July-August) produced by


Cyclotella caspia, in Mamaia Bay. Huge quantities 

of adult gobies, sole, plaice and turbot juveniles 

were washed up on the beaches or caught in 

lethargic condition from the sea by fishermen. 

As a matter of fact, HALLEGRAFF (1995) 

considers that species vegetating in densities over 

5∙10 6 cells∙l -1 are harmful, since phytoplankton 

hyperproduction leads to regular violations of the 

ecosystem carrying capacity and severe economic 

losses to aquaculture, fisheries and tourism 

operations [9]. 

However, some of algal species widely 

distributed at the Romanian coastal waters, such as 

Chaetoceros socialis, C. curvisetus, Dichtyocha 

speculum, Ceratium fusus, can seriously damage 

fish gills, either mechanically or through 

production of hemolytic substances. Other ones, 

such as P. minimum, Dinophysis acuta, 

D. acuminata, D, sacculus, D. rotundata, 

M. aeruginosa are considered potentially toxic 

species, having the capacity to produce potent 

toxins, like DSP (diarrheic shellfish poisoning), 

that through the food chain could cause a variety of 

gastrointestinal illness to humans [9]. 

The relationship between anthropogenic 

activities and changes in phytoplankton 

composition and diversity is one of the main 

objectives proposed in Harmful Algal Blooms 

research. Long time series of phytoplankton 

community storage in the NIMRD data base should 

be reconsidered related to HAB increase. 

5. References 

[1] BODEANU N., RUTA G., 1998 – Development 

of the planktonic algae in the Romanian 

Black Sea sector in 1981- 1996. In 

Harmful Algae, B. Reguera, J.Blanco, 

L.Fernandez, T. Wyatt (ed.) Vigo, Spain, 

1997: 188-191. 

[2] OGUZ T., DIPPNER J.W., KAYMAZ Z., 

2006 – Climatic regulation of the Black Sea 

hydro-meteorological and ecological 

properties at interannual-to-decadal time 

scale. Journal of Marine Systems, 6: 235- 

254. 

169 

[3] BODEANU N., 1987/1988 - Structure and 

dynamics of unicellular algal flora in the 

Romanian littoral of the Black Sea. Cercetari 

Marine, 20–21: 19–250. 

[4] MOROZOVA-VODIANITKAIA N.V., 1954 - 

The Black Sea phytoplankton, Tr. Sevastopol. 

Biol., 8: 11-99 (in Russian). 

[5] BSC, 2008. State of the Environment of the 

Black Sea (2001-2006/7). Edited by Temel 

Oguz. Publications of the Commission on the 

Protection of the Black Sea Against Pollution 

(BSC) 2008-3, Istanbul, Turkey: 23-49. 

[6] SKOLKA H.V., 1967 – Consideraţii asupra 

variaţiilor calitative şi cantitative ale 

fitoplanctonului litoralului românesc al Mării 

Negre. Ecologie Marină, Vol. 2: 193-293. 

[7] BODEANU N., ROBAN A., USURELU M., 

1981 – Elemente privind structura, dinamica şi 

producţia fitoplanctonului de la litoralul 

românesc al Mării Negre în perioada 1972- 

1977). Producţia şi productivitatea 

ecosistemelor acvatice. N. Botnariuc ed., Ed. 

Acad. Rom., Bucureşti: 42-50. 

[8] BODEANU N., ANDREI C., BOICENCO L., 

POPA L.,. SBURLEA A, 2004 – A new 

trend of the phytoplankton structure and 

dynamics in the Romanian marine waters. 

Cercetari Marine, 35: 77-86. 

[9] VELIKOVA V., MONCHEVA S.,. PETROVA 

D, 1999 – Phytoplankton dynamics and red 

tides (1987-1997) in the Bulgarian Black Sea. 

Wat. Sci. Tech., Vol. 39, No. 8: 27-36.


ASPECTS REGARDING THE BIODIVERSITY OF 

THE AQUATIC AND SEMI AQUATIC HETEROPTERA IN THE LAKES 

SITUATED IN THE MIDDLE BASIN OF THE OLT RIVER 

Daniela Minodora ILIE 

“Lucian Blaga” University, School of Sciences, Departament of Ecology and Environmental Protection, 

5-7 Dr. I. Raţiu Street, 550012, Sibiu, Romania 

__________________________________________________________________________________________ 

Abstract: The present work analyzes the bio diversity of the aquatic and semi aquatic heteroptera belonging to 

four habitats, respectively lakes situated within the middle basin of the Olt River. From the collected biological 

material, consisting of 724 samples there were identified 20 species of aquatic and semi aquatic heteroptera. We 

want to mention the presence of the species Paracorixa concinna in the lake in Cincşor, here being the single and 

only one appearance of this species till now in the basin of the Olt River. The different conditions of the 

researched habitats are to be seen in the structure of the communities of aquatic and semi aquatic heteroptera. 

The similitude among the established communities is a quite a reduced one. 

Keywords: aquatic and semi aquatic heteroptera fauna, communities analysis, the middle basin of the Olt 

__________________________________________________________________________________________ 


The aquatic and semi aquatic heteroptera lives 

in a great variety of habitats, from temporary swamps 

to big lakes, from brooks to small and big rivers, from 

continental waters to the surface of the oceans. The 

aquatic and semi aquatic heteroptera are consumers 

of the 2 nd degree (the food base consisting of both 

dead and alive prey). 

The present work proposes to evaluate the bio 

diversity of the communities of aquatic and semi 

aquatic heteroptera from the researched lakes that are 

situated in the following units of relief: Perşani 

Mountains, Făgăraş Mountanns and Hârtibaciu 

Plateau. The lakes are presented as follows: 

SO1: Bottomless Lake (Mateiaş) 

The lake is situated in Perşani Mountains, 

having the following coordinates: 45 0 59’ 08’’ N, 25 0 

20’ 

20’’ E, at an altitude of 522m. It is to be found on 

the left slope of the Olt River, being placed in the 

storages of the terrace allowing in this way the supply 

of the lake from the phreatic water. 

The lake, having a surface of approximately 

870m 2 

is surrounded by willows. Phragmites 

communis and Typha latifolia covers about 5% from 

the banks area. The vegetation above and under the 

water is about 55-60% of the surface of the lake. 

There are to be found Lemna minor, L. trisulca, 

Spyrogyra sp., and also Ceratophyllum demersum, 

Myriophyllum spicatum (a little). It is interesting to 

be mentioned the appearance in this station of the 

species Sagittaria latifolia and Potamogeton lucens 

that are seldom met in the middle basin of the Olt. 

SO2: Bâlea Lake 

The geographic coordinates are: 45 0 36’ 10’’ N, 

24 0 36’ 49’’E, at an altitude of 2036m. 

It is a typical glacier lake sheltered in the so 

called Bâlea bucket, nearby the separating limit 

between the glacial circle and the former glacial 

valley. There is a mixed supply, this being the spring 

of the river Bâlea. 

The lake has a surface of 4.6 ha and a maximum 

depth of 11.35. 

S03: Cincşor 

The lake is situated in the Hârtibaciu Plateau, 

having the geographic coordinates as follows: 45 0 49’ 

36’’ N, 24 0 49’55’’ E, at an altitude of 422m. It is an 

abandoned meander of the Olt River, which when 

there are big flood keeps in touch with the actual 

course of the river, being situated in its major 

riverbed. The supply of the lake is both from 

underground as well as superficial. 


Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) 

The banks of the river are covered with willows 

(Salix alba, S. triandra, S. fragilis), which make the 

banks more stable and also give shadow to the water. 

SO4: Netuş 

The Netuş Lake is situated in the Hârtibaciu 

Plateau having the coordinates as follows: 46 0 03’ 

55’’ N, 24 0 47’ 55’’ E, at an altitude of 484m. 

The lake was arranged by people, its purpose 

being to reduce the flood. It also has a fish breeding 

interest, being placed in the major riverbed of the 

Hârtibaciu River. The vegetation is undeveloped. 


The biologic material was gathered during 

September-October 2001, September 2002, August 

and September 2004, From three stations there were 

gathered two samples: in September and October 

2001 from SO!, in September 2002 and August 2004 

from SO2, in September and October 2001 from 

SO3. From the station SO4 there was done only one 

gathering (October 2004). For the identification of 

the species we used the determination key of the 

following authors: [1], [2], and [3]. 

There was calculated the relative abundance of 

each and every species from the researched habitats, 

diversity indexes ά - Margalef (for general aspects, 

such as the number of species and the number of 

individuals) and Lloyd-Ghelardi (for the evaluation of 

heterogeneity) – and the indicator of percentage 

similitude Renkonen, in accordance with [4]. 


As a result of the gatherings done during the 

periods mentioned before we identified a number of 

20 species, from which 13 species are aquatic 

heteroptera (Heteroptera: Nepomorpha) and 7 species 

are semi aquatic heteroptera (Heteroptera: 

Gerromorpha), belonging to 9 families, presented in a 

number of 724 samples (table 1). 

The Corixidae family is the best represented 

taking into account the number of species (8 species), 

but considering the number of the gathered 

individuals the Naucoridae family is on the first place 

(202 samples). At Mateiaş (SO1) we identified 17 

species representing 50% from the total number of 

species that were gathered in the middle basin of the 

172 

river Olt (Ilie, 2009). Here is the only station where 

appeared the species Ilyocoris cimicoides, its 

presence being linked to the under water vegetation. 

Other species of aquatic heteroptera (Sigara striata, 

Sigara iactans, Notonecta glauca, Plea minutissima) 

are also well represented from the same reason. On 

the other side, the vegetation above the water is 

favorable for the semi aquatic species (Microvelia 

reticulata, Mesovelia furcata and Mesovelia 

vittigera). 

The community of the aquatic and semi aquatic 

heteroptera from Mateiaş is defined by high values of 

the relative abundance of the species Ilyocoris 

cimicoides (A=30.31%), Microvelia reticulata 

(A=20.85%), Gerris argentatus (A=16.62%) and 

Sigara striata (A=12.54%) and values less than 10% 

for the other species. There is to be noticed an 

equilibrate structure of the heteroptera community as 

two species of aquatic heteroptera and respectively 

two species of semi aquatic heteroptera represents 

about 40% from the total of the community. On 

assembly the aquatic heteroptera represent 60% and 

the semi aquatic heteroptera about 40% from the 

heteroptera community in the lake in Mateiaş (in the 

terms of relative abundance). The species Notonecta 

glauca is represented by an average number of 

individuals, the dimensions of the population being 

determined by the big size and the predator behavior, 

which is extremely active. 

At the Bâlea Lake (SO2) we identified only two 

species of heteroptera although there was done the 

some number of gatherings, the habitat being of the 

same kind (natural lake) and the relief unit the same, 

namely mountain. This fact is a result of the great 

differences of altitude, which implies climate 

differences (especially the temperature, on which 

depends the existence and the development of the 

insects) as well as the vegetation (this being mainly a 

shelter against the predators). There was also noticed 

the fact that the species that were present in the Bâlea 

Lake are to be found in the Mateiaş Lake, too. 

At Cincşor (SO3) there were identified 10 

species of aquatic and semi aquatic heteroptera. The 

most of the species belong to Corixidae family (4 

species). The other families are represented by one or 

maximum two species. 

In the aquatic and semi aquatic heteroptera 

community of the Cincşor Lake, Micronecta scholtzi

Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) 

registered by far the highest value of the relative Table 3. The values of the Renkonen index 

abundance (A=46.00%). Some authors considered 

important the fact that fish eat several species of 

heteroptera, especially Corixidae, reducing in this 

way their populations [5]. The populations of the 

species Micronecta scholtzi were noticed in the shore 

S01- S01- S01- S02- S02- S03- 

S02 S03 S04 S03 S04 S04 

R. 23.87 19.40 21.00 0.00 22.22 0.00 

The similitude between the aquatic and semi 

aquatic heteroptera communities in the 4 habitats was 

area of the aquatic habitat, a shadowed area and 

without underwater plants, having a reduced depth, 

which is not favorable for fish, this being the 

explanation of the abundance of this species in the 

heteroptera community. We also want to notice the 

presence of the species Paracorixa concinna, here 

being the only once it was registered till now in the 

basin of the Olt River [6]. 

At Netuş (SO4) there were identified two 

species of semi aquatic heteroptera: Gerris lacustris, 

being collected 7 individuals and Microvelia 

reticulata, 2 individuals being collected. In this case 

the number of species is a much reduced one because 

the ecologic conditions are not proper for these 

heteroptera. We want to notice that both species are 

semi aquatic ones, these being less sensitive than the 

aquatic species regarding the volume and the density 

of the underwater as well as the floating vegetation. 

Table 2. The values of the diversity indexes ά 

obtained for every collecting station 

Index / Station S01 S02 S03 S04 

Margalef 2.46 

3 

Lloyd-Ghelardi 0.68 

6 

0.91 

0 

0.91 

8 

2.30 

1 

0.71 

3 

0.45 

5 

0.76 

4 

The values of Margalef index are quite high for 

the habitats SO1 (2.463) and SO3 (2.301) 

respectively low for the habitats SO2 (0.910) and 

SO4 (0.455) (table 2). The higher values of the index 

show that there were better conditions in the habitat 

for the heteroptera species. 

The Lloyd-Ghelardi index, varying between 0 

and 1, shows for the researched habitats a relatively 

homogenous repartition of the individuals on the 

species, representing around 70% of the optimum 

value. SO2 is an exception having a higher value 

because of the identification of individuals number 

closed to the species number. 

173 

established having as a base the Renkonen index, 

calculated with data of relative abundance of the 

species. It came out that there was a quite low 

similitude (table 3). 


There were identified 20 species, from which 

we noticed the species Paracorixa concinna, at 

Cincşor being the only registration in the basin of the 

Olt River. 

The number of the identified species in every 

habitat differs quite a lot (among 2-17 species) as 

well as the abundances of different species within the 

communities that establish them in those habitats (for 

example in SO1, the only station where the species 

Ilyocoris cimicoides was present, this being also the 

most abundant; in SO3 Micronecta sholtzi registered 

by far the highest value of the relative abundance). 

These show the variety of the conditions that are 

existent in those lakes; for the aquatic and semi 

aquatic heteroptera the quality of the habitats is 

connected with the altitude, damming, the 

development of the aquatic vegetation, the fish 

population, etc. The similitude between the 

communities of aquatic and semi aquatic heteroptera 

established in those 4 habitats is quite a low one. 

5. References 

[1] DAVIDEANU, ANA, 1999. Contribuţii la studiul 

heteropterelor acvatice din România, Teza de 

doctorat, Univ. ”Al. I. Cuza”, Iaşi, 427 pp. 

[2] JANSSON, A., 1986. The Corixidae (Heteroptera) 

of Europe and some adjacent regions, Acta Entom. 

Fennica, 47: 1-92. 

[3] POISSON, R., 1957. Hétéroptères aquatiques 

(Faune de France), 61: 1-263. 

[4] SÎRBU, I., BENEDEK ANA MARIA, 2004. 

Ecologie practică, Univ. Lucian Blaga, Sibiu, 1- 

264.


[5] PAPÁČEK, M., 2001. Small aquatic and ripicolous 

bugs (Heteroptera: Nepomorpha) as predators and 

prey, Eur. J. Entomol., 98: 1-12. 

[6] ILIE, DANIELA MINODORA, 2009. 

Heteropterele acvatice şi semiacvatice din 

bazinul mijlociu al Oltului, Ed. Altip, Alba-Iulia, 

1-279. 

174

Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010) 

Table 1. The identified species of aquatic and semi aquatic heteroptera from the researched habitats and the 

values of the relative abundance 

Gathering Station S01 S02 S03 S04 

Taxon 

Individuals 

number 

A% Individuals 

number 

174 


number 


number 

Fam. Gerridae 

Gerris argentatus 110 16,62 5 10,00 

Gerris 

odontogaster 3 0,45 

Gerris lacustris 1 0,15 7 77,78 

Fam. Veliidae 

Microvelia 

reticulata 138 20,85 1 33,33 2 22,22 

Fam. 

Hydrometridae 

Hydrometra 

stagnorum 1 0,15 11 22,00 

Fam 

Mesoveliidae 

Mesovelia furcata 21 3,17 4 8,00 

Mesovelia vitigera 10 1,51 1 2,00 

Fam. Corixidae 

Micronecta 

(Dichaetonecta) 

scholtzi 23 46,00 

Corixa punctata 6 0,91 

Hesperocorixa 

linnaei 2 0,30 

Paracorixa 

concinna 1 2,00 

Sigara 

(Retrocorixa) 

limitata 1 0,15 

Sigara (Sigara) 

striata 83 12,54 

Sigara (Subsigara) 

iactans 45 6,80 1 2,00 

Sigara 

(Vermicorixa) 

lateralis 1 2,00 

Fam. Naucoridae 

Ilyocoris 

cimicoides 202 30,51 

Fam. Nepidae 

Nepa cinerea 2 0,30 1 2,00 

Fam. 

Notonectidae 

A%


Notonecta viridis 2 0,30 

Notonecta glauca 

Fam. Pleidae 

20 3,02 2 66,67 

Plea minutissima 

Individuals 

15 2,27 2 4,00 

number per 

662 3 50 9 

gathering station 

Species number 

per gathering 

station 

17 2 10 2 

175


PROGRAM OF PREVENTION AND CONTROL OF FUNGUS INFESTATION OF 

GRAIN AND FODDER , HUMAN AND ANIMAL PROTECTION AGAINST 

MYCOTOXINS 

drd.ing. Ioan Aurel POP*, conf. dr. Augustin CURTICĂPEAN**, drd.ing. Alin GULEA*, dr. Cornel PODAR*, 

ing. Iustina LOBONTIU*. 

* Staţiunea de Cercetare Dezvoltare penru Creşterea Bovinelor Mureş, 

str. Principală 1227, Sângeorgiu de Mureş, jud Mureş. 

** Universitatea de Medicină şi Farmacie Târgu Mureş 

__________________________________________________________________________________________ 

Abstract: mycotoxins contained in forages may yield to cause different health issues on farm livestock as 

decreasing the forage intake and bioconversion, serious illness and death. Food and Agriculture Organization 

(FAO) appreciates on global level that 25% of agricultural products are contaminated with mycotoxins. These 

compounds contaminate feeds before and after harvesting. Food quality monitoring on each stage, especially due 

to it’s fungal potential risk is very important for the development of antifungal strategies adapted to local 

conditions. Thus, through a research project witch involves the quantification of mycotoxins concentrations from 

feed and food samples taken from different farms located in Central Region of Transylvania we managed to 

develop a new method of detection and quantification of three mycotoxins. The paper work presents a part of 

activities performed in a research project and comprises their results on preventing and control of funguses and 

mycotoxins. 

Keywords: mycotoxins, fungus, crops, methods. 

__________________________________________________________________________________________ 


Food safety has become one of the directions 

very important area to protect and improve the 

quality of life. To ensure all elements contributing to 

the increase of consumer protection and food quality, 

develop new methods of control, as simple, low 

resource consuming, while used in normal conditions 

[1]. Thus, eliminating sources of toxic advanced 

occurring in food composition is a major goal. One 

such source is the species of fungi producing 

mycotoxins, which are found in most foods of plant 

origin whose storage / storage is inadequate, but 

worse is that we find and their metabolites in animal 

products, products from infested feeding. 

Monitoring primary storage conditions, and 

assessment on a representative sample of infestation 

by specific analysis will recommend specific methods 

of prevention / treatment of developing adverse 

effects of mycotoxins in crops. 

In a research project has developed a new 

method for detection and quantification of three 

mycotoxins for monitoring the infection status of feed 

and food grain with mycotoxins in various units and 

areas located in Region Development Centru. [2] 

The paper also presents results of experiments: 

- Study the behavior of wheat, barley, triticales 

and corn hybrids tested in comparative culture from 

the years 2008, 2009 from SCDCB Mures and their 

hierarchy according to their resistance to disease 

attack; 

- Testing of eight plant protection products for 

disease prevention and control in cereals in climatic 

conditions in 2009 and monitoring the behavior of 

fungicides in the production. 


Thurough the research project "Complex 

program of prevention and control of fungus 

infestation for grain and fodder for providing animal 


Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) 

wealth and consumer protection’ has achieved a 

status of monitoring infestation of feed and food 

grain with mycotoxins in various units and sectors 

located in the Development Region Center. Action 

was initiated in early June in a maximum period of 

susceptibility to the incidence of mycotoxins deposit 

being made by the team of researchers from SCDCB 

Mures Tg Mures and SC AGROFITOPLANT 

PharmacyLtd. 

Sampling activity was made taking account of 

Regulation (EC) NO. 401/2006 laying down the 

procedures for sampling and analysis methods for 

official control of mycotoxins in food. 

In total 180 samples were taken from 44 production 

units, which have in operation a total: 14 450 ha of 

arable land, 8960 cattle, 29,170 porcine, 36,640 

birds, 8792 sheep. Average area of farms covered 

operating is 328 hectares. 

Of the total samples: 145 samples were 

concentrated (maize grain, flour, PVM's, milk 

powder) and 35 samples of forage (silage, half hay, 

hay, grains, beet chips, etc.). [1] 

The quantification of mycotoxins in the 

samples, at the UMF Mures (Mures University of 

Medicine and Pharmacy) has developed a new 

method to quantify the simultaneous separation of 

mycotoxins by liquid chromatography HPLC using a 

DIONEX Ultimate 3000 with UV detection 

simultaneously on different channels. Optimization 

method was performed to determine simultaneously, 

using an ordinary system, more relevant mycotoxins 

present in samples of feed corn stored for eight 

months. 

Based on production and behavior have 

established multi fenophasic comparative cultural 

components subject to this project, DC M01 and M02 

with wheat varieties, triticale and barley. (Table 1) 

Table 1. Crop ranking regarding yield DON and 

ZON. 

Specia/Soiu 

l 

Producti 

a medie 

(Kg/ha) Isc 

Ierarh 

i 

zare 

I GRAU 6920 

Ariesan(Mt 6780,9 120,20 V 

7243,5 161,01 I 

178 

II 

Ardeal 1 

Magistral 7159,1 130,59 III 

Renan 6536,0 114,56 VI 

Exotic 7047,9 133,56 II 

Gasparom 7070,5 122,90 IV 

Turda 

14/98 6671,0 105,98 VIII 

Apullum 6851,8 110,33 VII 

TRITICAL 

E 7699 

Plai(Mt) 7798,7 120,20 III 

Titan 7971,4 123,85 II 

Trilstar 7501,8 104,89 V 

Stil 7590,3 117,51 IV 

00474T1-1 7632,1 125,30 I 

II ORZ 6481 

Gerlac(Mt) 6570,8 110,00 I 

Regal 6683,2 97,12 III 

Plaisant 6189,1 98,81 II 

For testing resistance to major pests and 

diseases of maize hybrids grown in the area was 

established in late April (2008.2009) a crop of corn 

hybrids compared with 24 (S = 1000 m) in the 

experimental field of the resort located in Sg Mures. 

The main observations made: plant vigor, flowering 

time, date of silk, drought resistance, 

Helminthosporium sp., Puccini sp., Ustillago sp. 

Attack of Fusarium sp., The number of sterile plants, 

the number of broken and fallen plants, resistance to 

attack pest and grain production. 

The content determination of mycotoxins was 

performed at UMF Targu Mures (University of 

Medicine and Pharmacy. Mures) SPC Mures (Mures 

Public Health Center) Promovert laboratories in 

Champagne, France (company Bayer). [1] 


Precision method for determining meets the 

minimum relative standard deviation (with values in a 

field of ± 15%) for quality control samples measured

Ioan Aurel Pop et al. / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) 

in both samples the same day and comparisons 

between samples from different days. 

Minimum limits of quantification for the three 

analytes / mycotoxins (2.88 ng / mL AflaB1, 2.88 ng 

/ mL respectively OchrA 14.4 ng / mL Zeara) met the 

requirements of precision and accuracy so that the 

relative standard deviation to be included in a field ± 

20% for both measurements on the same day as well 

as those performed on different days and the 

difference between mean calculated and nominal 

values (BIAS%) is also contained in a maximum field 

of ± 20%. 

Records on the evidence provided by corn and 

also on samples from various forage plants show an 

infestation of their importance to all three classes of 

mycotoxins, so Aflatoxin B1 and Ochratoxin A with 

zearalenone. Infestation levels are relatively high, 

regulated levels overruns are much more frequent and 

more significant if the first two Mycotoxins - 

Aflatoxin B1 respectively Ochratoxin A. Thus, the 

calculated values for concentrations of mycotoxin 

present in almost all unknown samples analyzed 

exceed permissible concentrations and regulated at 

European level. [2] 

High levels of mycotoxins found in animal feed 

and is probably due to the chosen period when this 

work started in early June, during which cereal stocks 

are running, close grain with a one year old storage 

warehouses before the process is Cleaning for storing 

grain harvest. 

Using production data obtained, the 

observations on different varieties fenophase attack 

on disease resistance, deoxynivalenol and 

zearalenone content in samples taken at harvest 16 

varieties of cereals were ranked using a synthetic 

index calculated Isc this. 

Results show that there are differences between 

varieties in terms of mycotoxins but not the values 

obtained exceeding the maximum allowed by law 

(1250 ng / g DON, and 100 ng / g Zon). 

Maize, based on production, moisture at 

harvest, percentage of plants broken and fallen and 

observations of vegetation during the attack on 

disease resistance of a synthetic index was calculated 

to ease the process generally ranking of cultivars. 

Results of tests carried out in laboratories SC 

Bayer SRL Promovert in Champagne, France 

reinforce the lessons learned so far. Of the seven 

179 

variants examined only version control - untreated 

with fungicide containing deoxynivalenol was above 

the limit of quantitation of 220 ng / g, respectively 

440 ng / g value in joining the legal permissible limit 

of 1250 ng / g DON. [1] 

% spice sanatoase 

101 

100 

99 

98 

97 

96 

95 

94 

93 

92 

95 

Martor 

netratat 

100 100 

Folicur Solo 

250 EW 

97 

100 

Tilt 250 EW Duett Ultra 

Produsul 

Prosaro 250 

EC 

97 97 

Nativo 300 

SC 

Falcon 460 

EC 

Fig. 1. Fungicides effect in Fusarium removal 

from Ariesat wheat variety at Tg Mures. 


Interpreted data show that the current 

methodology for preparing samples for analysis / 

quantification of mycotoxin content of substances of 

category has limits too generous. Thus, extraction of 

these substances (of which there are complex 

matrices) respecting the standardized methods, shows 

a lower sensitivity, which leads to highlighting of 

quantities / concentrations lower than actual. .[2] 

The existence of evidence over the maximum 

levels allowable by law certify the importance of this 

research and the need for a regional research 

antimycotic. 

Climatic conditions of the agricultural year 2008 

- 2009, characterized by high temperatures 

throughout the crop growing season and low rainfall 

than -114 mm limited attack foliar and ear diseases, 

and the effect of crop protection products was not 

very visible. For further research would require more 

years of study to catch different climates. 

Large assortment of hybrid corn study allows 

farmers to select hybrids with high production 

potential and adaptability to the conditions of the 

area. To limit the attack of diseases and in particular 

Fusarium in seed must be transmitted primarily by 

limiting attack Pyrausta which facilitates infection

Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010) 

how damaging fungal diseases of plants and causes 

breaking of preventing deployment of mechanized 

harvesting in good condition. [3] 

5. References 

[1] POP I., GULEA A., CURTICĂPEAN A., 

PODAR C. , 2009 - Program complex de 

prevenire şi combatere a infestării cu miceţi la 

cereale şi plante furajere pentru asigurarea 

bunăstǎrii animalelor şi protecţia 

consumatorilor, Raport de progres Transa a II-a. 

[2] A. CURTICĂPEAN, FELICIA TOMA, 

MONICA TARCEA, MANUELA 

CURTICĂPEAN, VICTOR SĂMĂRGHITAN, 

I. POP, A. GULEA, 2009 - Optimizarea unei 

metode HPLC de separare şi determinarea 

simultană a unor micotoxine din porumb,- Noi 

tendinţe şi strategii in chimia materialelor 

avansate. Institutul de Chimie Timişoara, 

Timişoara. 

[3] Pop I., Gulea A., Curticăpean A., Podar Cornel, 

2009 - ‚Program complex de prevenire şi 

combatere a infestării cu miceţi la cereale şi 

plante furajere pentru asigurarea bunăstǎrii 

animalelor şi protecţia consumatorilor’- Raport 

de progres Transa I. 

180


DATA ON THE DYNAMICS OF SOME MICROBIAL GROUPS IN SOILS 

WITH DIFFERENT TROPHIC STATUS IN CUMPĂNA REGION (DOBROUDJA) 

Elena DELCĂ 

Ovidius University of Constanţa, Faculty of Natural Sciences and Agricultural Sciences 

Mamaia Avenue, No. 124, Constanţa, 900527, Romania, 

Doctoral school Biology, Specialization Ecology 

__________________________________________________________________________________________ 

Abstract: The aim of paper was to assess the effect of administration of organic amendments on the dynamics of 

the abundance of microbial groups significant in nutrient cycling in soils. Abundance of total culturable bacteria 

ranged from 19.93x10 6 UFC/g dry soil, to 501.79x10 6 UFC/g dry soil. When soil was supplemented with manure 

microbial density showed a significant increase 501.79x10 6 UFC/g dry soil compared with control variant. 

Bacterial density increased significantly as value, too, following the administration of specific biofertilizers 

(Biovin, Bactofil Professional; Mycos Green), up to 142.13 x106UFC/g dry soil. Inorganic fertilizers did not 

have a positive effect on microbial density values, being more or less similar to those reported for the control. 

Our preliminary data show that organic amendments with complex composition have a direct effect on the 

abundance and diversity of soil and influence indirectly the microbial metabolism and nutrient cycling rate. 

Keywords: humus, microorganisms, bioactivators, fertility 

_________________________________________________________________________________________ 


To start and propose a suitable biological soil 

reconstruction plan it was necessary to initiate a 

series of observations and experiments in a 

characteristic agroecosystem of Dobroudja (Cumpana 

commune) in order to assess the current biological 

status. Using new agricultural technology, and adding 

different fertilizers the experiments have the aim to 

improve the number and activity of soil 

microorganism and indirectly to enhance the rate of 

organic matter decomposition. This would improve 

over time the soil structure and restore the stock of 

humus in the soil. 


The experiments have taken place on a 7.5 ha 

plot situated in the outside of Cumpana, in Constanta 

district. Josef wheat was cultivated on the entire area, 

which was divided in 7 variants, each variant being 

administered a different type of fertilizer in different 

quantities and periods, as follows: 

- Variant I - only chemical fertilizers - 100kg/ha 

N15P25K15 in autumn, 150kg/ha NH4NO3 at the 

beginning of spring; 

- Variant II – Biovin organic fertilizer 400kg/ha and 

Biovin 30 of l/ha, ½ at herbicide stage and ½ at flour 

stage; 

- Variant III – garden soil - 15t/ha in autumn; 

- Variant VI – l/ha of Biovin 30, ½ at herbicide stage 

and ½ at flour stage; 

- Variant V – Biovin 150kg/ha administered during 

sowing, 150kg/ha NH4NO3, 40kg/ha at the beginning 

of spring, 50kg/ha at herbicide stage and 60kg/ha at 

flour stage; 

- Variant VI – Biovin 375kg/ha, liquid Biovin 30 of 

l/ha, ½ at herbicide stage and ½ at flour stage, 1mc 

Green Mycos, 1l Bactofil Professional; 

- Variant – March – were not applied amendments. 

Biovin Fertilizers are being administered for the 

first time in Dobrogea. 

Biovin is being produced through a 

technological process from grape kernels. 12 years of 

western research proved the following: it aerates the 

soil, improves it (it contains up to 70% humus 

makers), and purveys all plants with nutritive 

elements and biostimulators, it enriches the soil with 


Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) 

microorganisms that create humus, it strengthens the 

roots and it multiplies the percentage of smooth roots 

and radicular wintergr; 

Bactofil Professional is a product for 

improving the soil biological quality and contains 

nitrogen fixing bacteria phosphate-solubilization 

bacteria, and heterotrophic bacteria that stimulates 

the decomposition of organic matter. 

Green Mycos is a product containing 

arbuscular mycorrhizal fungi and a number of factors 

that stimulate the establishment of symbiosis, 

improving the soil quality up to 20 years. [1] 

Experiments began in autumn 2009 by sampling 

the soil at a depth of 15 cm approximately, followed 

by quantification of some agrochemical (humus, 

indice N2) and biological parameters (bacteria, free 

N2-fixing bacateria, actinomycetes, microfungi). 

Quantitative determination of microbial 

abundance was done by decimal dilutions of soil 

followed by inoculation of known quantities on solid 

nutrient media. For this purpose, after weighing the 

samples were inoculated on culture medium with a 

specific composition. Thus, to determine the number 

of total culturable heterotrophic bacteria it has been 

used nutrient: 

- agar medium [2], [3] [4] - (pulvis yeast extract 

2.5 g, peptone 0.2 g, Agar 17-20g. It was sterilized 

20 min at 120 o C); 

- free N2-fixing bacteria on Ashby medium, [5] 

(15g Mannitol; g K2HPO4 0.2, MgSO4 ∙ 7H2O 0.5 g, 

0.2 g NaCl, CaSO4 ∙ 7H2O 0.1 g, CaCO3 5g, Agar 

17-20g. was sterilized 30 min at 115 o C). 

Determination was made on the environment 

actinomicete Czapeck – Dox ( 3g NaNO3; 1g 

K2HPO4; 0,5g MgSO4 ; 0,5g KCl; FeSO4 traces; 

Sucrose 30g; 17-20g Agar; pH 5,5; it was sterilized 

30 min at 115 o C) [3], [4], [7] and the abundance of 

microfungi was determined on Sabouraud medium 

(CaCl2 0.5g, 0.1g K2HPO4, KH2PO4 0.1g, 10% 

MoO3 0.1ml, 0.05ml FeCl3 10%, was sterilized 30 

min at 115 o C). 

The total number of bacteria per gram of soil 

was calculated using the formula: no. bacteria, 

actinomycetes, microfungi = X colonies x dilution x 

10 x 100/100-U where X = average of colonies 

grown on culture medium, 10 = balancing coefficient 

of 0.1 ml of inoculum in the reporting of dilution soil 

U% = soil moisture. [8] 

182 


The initial estimations have revealed a relatively 

low abundance variability between different 

experimental variants. 

Thus, the lowest abundance was detected in 

variant VI, heterotrophic bacteria having a mean 

abundance of 19.93 x 10 6 CFU/g dry soil (Fig. 1). 

The abundance was highest instead on variant 

II, in which case the total number of heterotrophic 

bacteria reached 45.45 x 10 6 CFU / g dry soil (Fig. 

1). 

Fig. 1 Bacterial density distribution in the initial 

stage of the experiment (October 2009) 

Changes in microbial abundance in 

experimental and control reflect the heterogeneity of 

normal physicochemical and trophic conditions of the 

soil, the values recorded can be considered normal 

for chernozem soil type. 

Fig. 2 Distribution of heterotrophic bacterial density 

after six months of application of amendments (May 

2010)

Elena Delca / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) 

After six months of application of organic and 

inorganic amendments microbial abundance showed 

considerable changes in some cases, so the highest 

density was recorded in the experimental group 

fertilized with manure, variant III, in which case we 

determined a density of 501.79 x 10 6 CFU/g dry soil 

(Fig. 2). The number of bacteria also increased 

significantly in variant VI up to 142.13 x 10 6 CFU/g 

dry soil (Fig. 2). 

Paradoxically, after six months I have noticed 

decrease of heterotrophic bacteria in variant I, which 

might be explained by the effect of administration of 

chemical fertilizers and organic substance 

consumption by bacteria. In autumn 2009 the initial 

amount of organic matter in the form of crop residue 

remaining after harvest decreased gradually as the 

decomposition and microbial consumption 

progressed and provide sufficient nutrients to 

maintain viable bacterial population as numerous as 

in the beginning of the experiment. In other variants 

(II, IV and V) microbial density presented weakly 

peaks compared with the control, and ranged between 

30.73 x 10 6 CFU/g soil dry and 46.31 x 10 6 CFU/g 

soil dry (Fig. 2), situation observed also in control 

variant. 

At the beginning of the experiment, density of 

free nitrogen-fixing bacteria was relatively low, (Fig. 

3), ranging from 10.3 x 10 5 CFU/g dry soil to 26.7 x 

10 5 CFU/g dry soil. In case of variants I, III, V and 

VI values are very close to those recorded for control 

(Fig. 3). 

Fig. 3 Changes of abundance of nitrogen-fixing 

bacteria (October 2009) 

183 

The highest number recorded for variants II and 

IV ranging between 22.7 x 10 5 CFU/g dry soil and 

26.7 x 10 5 CFU/g dry soil, rely on local trophic 

conditions. 

In any case, there was a certain uniformity of 

abundance of nitrogen-fixing bacteria beginning of 

the experiment. At this stage relatively low 

abundance of nitrogen-fixing bacteria could be due to 

higher quantity of organic substance that stimulates 

competition within heterotrophic bacterial 

populations. 

Fig. 4 Change in binding density micro N2, after six 

months (May 2010) 

After six months of adding the fertilizers our 

estimations revealed a significant increase of the 

number of nitrogen-fixing bacteria, including that of 

the control (Fig. 4). 

Most evident increase of abundance of this 

group occurred I in variant III, where we recorded 

about 195.81 x 10 5 CFU/g dry soil (Fig. 4). 

Significant increases were recorded in the case 

of variants II, IV and VI, where abundance ranged 

from 90.44 x 10 5 CFU/g dry soil to 127.39 x 10 5 

CFU/g dry soil (Fig. 4). 

In variant I and V, values were close to the 

abundance determined for control (Fig. 4). 

Table 1. Agrochemical analysis conducted in autumn 

2009 

Nr. 

Crt. 

Variant % mg/Kg 

Humus Indice N2 

1 V1 2.9 0.14

Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010) 

2 V2 2.95 0.14 

3 V3 3.21 0.15 

4 V4 3.07 0.15 

5 V5 2.83 0.13 

6 V6 3.09 0.15 

7 M1 3.16 0.15 

Table 2. Agrochemical analysis conducted after 6 

months (May 2010) 

Nr. 

Crt. 

Variant % mg/Kg 

Humus Indice N2 

1 V1 3.07 0.15 

2 V2 3.09 0.15 

3 V3 3.36 0.17 

4 V4 3.12 0.15 

5 V5 3.07 0.15 

6 V6 3.12 0.15 

7 M1 3.24 0.16 

Further information relative determination of 

humus were not noted substantial increases for the 

period under review (Table 1, Table 2), which is 

understandable due to the short observation time 

insufficient to identify significant changes in the 

humus content. 


Dynamics of the total number of heterotrophic 

bacteria presented significant changes after 

application of amendments. The most significant 

increase occurred in the variant enriched with 

manure, trend was also observed in the case of 

variant VI in which soil was treated with Biovin. 

The total number of nitrogen fixing bacteria 

showed a spectacular increase after six months of 

amendments application, effect that can be attributed 

only in part as a result of fertilizer. 

Results of soil chemical and microbiological 

analysis reveal a low contribution of microorganisms 

to the improvement of the soil fertility and microbial 

biodiversity. To improve the biological quality of soil 

it is necessary to increase the biomass of 

microorganisms in the soil by adding bacteria, and 

184 

their activities by introducing large amounts of 

organic matter. 

5. References 

[1] BERCA M, 2008 – Probleme de ecologia solului. 

Editura ceres, 2008: 43-63. 

[2] BERGEY’S, 1986 - Manual of Sistematic 

Bacteriology, vol. 2, Williams and Wilkins, 

Baltimore, USA, 4087: 1075-1079 

[3] CLARK F, 1965 - Agar plate method for total 

microbial count. Method for Soil Analysis, vol.2: 

1460-1465 Amercian Society for Agronomy, 

Madison, WL. 

[4] FLORENZANO G, 1983 - Fondamenti di 

microbiologia del rerreno, Reda Ed, Firenze, 630: 

115-136. 

[5] PAPACOSTEA P, 1976 - Biologia solului, Ed. 

Ştiinţifică şi Enciclopedica, Bucureşti, 272: 81- 

259. 

[6] PITT JL, 1991 - A Laboratory Guide to common 

Penicillium Species, USA, 184: 129-135. 

[7] TSUNEO WATANABE, 2001 - Pictorial Atlas of 

Soil and Seed Fungi, Morphologies of Cultured 

Fungi and Key to Species – Second edition, CRC 

Press, 504: 230-236. 

[8] DUMITRU M, TOTI M, VOICULESCU A-R, 

2005 – Decontaminarea solurilor poluate cu 

compuşi organici, Ed. Sitech, Bucureşti, 364: 

262-266.


THE AGRICULTURAL POTENTIAL OF PHOSPHOGYPSUM WASTE PILES 

Lucian MATEI 

Pescarusului Street, Bl CP1, Sc B, Ap 18, Navodari, Constanţa County, Romania, 

e-mail: mat_lucian@yahoo.com 

__________________________________________________________________________________________ 

Abstract: The cultivation of Salix sp. on the phosphogypsum waste piles started from the wish to discover a 

cheap, efficient, and ecological covering method. For this purpose, Salix alba and Salix fragilis cuttings were 

used, as they were collected from an area adjacent to the town of Navodari. Some Salix fragilis cuttings were 

collected from the trees that grew spontaneously on the waste piles. The species Salix alba is newly introduced in 

the ecosystem of the phosphogypsum waste pile. The species of the genus Salix are dioicous. As they are not 

fertile, S. alba and S. fragilis are often crossbred in nature, creating hybrids, the most popular being S. x rubens. 

The large number of hybrids of the genus Salix offers them increased capacity to adapt and exist in the most 

various environmental conditions. The purpose of the project is to identify a species or a hybrid that, given the 

life conditions on the phosphogypsum waste pile, should offer a considerable quantity of wood mass per ha in 

order to collect and exploit it as solid fuel. 

Keywords: Salix, phosphogypsum waste piles, ecological reconstruction, phytoreparation 

__________________________________________________________________________________________ 


The idea of cultivating Salix sp. on the 

phosphogypsum waste piles started from the wish to 

discover a cheap, efficient, and ecological covering 

method. 

The phosphogypsum waste pile number 3, which 

belongs to the S.C. Fertilchim – Marway S.A. 

company, is the result of massive accumulations of 

phosphogypsum obtained by the wet method of 

making phosphorus fertilizers. The waste pile is 

rectangular and has a surface of approximately 21 ha. 

In 1996, it was removed from the technological flux 

and a poor vegetation settled spontaneously on its 

surface over the next few years. A study regarding 

flora, accomplished in 2009, identified 35 species of 

plants [1]. The dominant species is Puccinellia 

distans, a grass that prefers salty soils (Poaceae) [2]. 

Apart from this dominant species, waste pile number 

3 also displays a mixture of various species in terms 

of preference for the environmental conditions. Thus, 

xerophile species such as Tamarix ramosissima live 

together with hygrophile species such as Salix fragilis 

and Salix matsudana. We can also encounter 

spontaneous Salix caprea (mesophile) on the 

phosphogypsum waste pile. 

The species of the genus Salix are dioicous, the 

sexes being separate: the trees bear male or female 

flowers. Some species of the genus Salix are 

interfertile. Different varieties of Salix alba and Salix 

fragilis crossbreed frequently in nature giving birth to 

different hybrids, among which the most common is 

Salix x rubens. The overlapping of the morphological 

features of the two species increases the degree of 

difficulty in the correct determination of the species 

[3]. Both the morphological studies and the genetic 

investigations realized on the S. alba – S. x rubens – 

S. fragilis complex indicate its division into two main 

groups. A group is made up of Salix alba and Salix x 

rubens, while the second group is made up of S. 

fragilis si S. x rubens var. basfordiana [4]. This 

division of the complex into the two groups concords 

with previous research (Triest et al., 1998, 2000), 

quoted by [4], who analyzed the isoenzymes and 

RAPD (Random Amplified Polymorphic DNA). As a 

result of these tests, the S. alba – S. x rubens – S. 

fragilis complex was divided into two groups: “S. 

alba-like” and “S. fragilis-like”. 


The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) 

The great variety of hybrids existing in nature 

prevents sometimes the exact identification of the 

species of the genus Salix only by morphological 

features. It is possible that the specimens identified 

on the phosphogypsum waste pile as belonging to the 

species S. fragilis, might be hybrids that inherited 

from the genitors the capacity to live on a salty 

substrate which lacks organic matter but has high 

humidity. Unfortunately, the lack of financial means 

prevented the accomplishment of ADNcp (ADN 

chloroplastic) analyses that allow the precise 

identification of the species or supposed hybrids used 

within the experimental project for the setting up of a 

willow culture. The large number of hybrids of 

species of the genus Salix offers them increased 

capacities to adapt to the most diverse environmental 

conditions. 

Starting from this theory, the experimental 

culture using species of the genus Salix on the 

phosphogypsum waste pile seeks to identify a species 

or a hybrid that, in the living conditions of the waste 

pile, should provide the most considerable quantity of 

wood mass. I mention that the species Salix alba is 

newly introduced in the ecosystem of the 

phosphogypsum waste pile with the purpose of 

monitoring the production of biomass reported per 

surface unit. 


The experimental culture of Salix sp. on 

phosphogypsum waste pile no. 3 belonging to the 

S.C. Fertilchim – Marway S.A. company was set up 

on a vegetation-free surface of 540 square meters. 

For this purpose, Salix alba and Salix fragilis cuttings 

were used. They were collected from an area adjacent 

to the town of Navodari. Some Salix fragilis cuttings 

were collected from the trees that grew spontaneously 

on the waste piles. This surface was planted with 110 

cuttings belonging to the species S. alba (44 pieces) 

and S. fragilis (6 pieces). The cuttings were planted 

on parallel rows with a length of 20 m. The distance 

between two successive rows is three meters, while 

the distance between the cuttings on the same row is 

two meters. The number of cuttings on a row is 

eleven. The planting depth is between 0.7 and one 

meter. Thus, two rows with S. fragilis were planted at 

186 

0.7 meters and eight rows (four with S. alba and four 

with S. fragilis) were planted at a depth of one meter. 

The collection of cuttings occurred between 10-20 

March 2010, while 

their planting took place between 16-27 March 2010. 

Some of the cuttings (22 pieces) were collected from 

S. fragilis grown spontaneously on the 

phosphogypsum pile, while the others (88 pieces) – 

44 S. alba and 44 S. fragilis – were collected from 

the area adjacent to the town of Navodari. The age of 

the cuttings is between one and three years, while 

their sizes vary between one and 2.5 meters. 

In order to verify the influence of the 

microclimate effect, five rows of cuttings were 

planted in phosphogypsum ditches. The depth of the 

ditch was approximately 0.4 meters, while the width 

was 0.3 meters. The planting depth in four of the five 

ditches was measured to be one meter, taking the 

phosphogypsum surface as marker and not one meter, 

taking the bottom of the ditch as marker. In the case 

of the fifth ditch, the planting depth is 0.7 meters and 

it was measured the same as in the previous rows. 

The planting method is presented in Figure 1. 

Fig.1. The way in which the cuttings were planted 

The other five rows that make up the witness 

area for the study of the microclimate influence were 

planted directly on the phosphogypsum surface, 

without digging ditches. The planting depth in the 

case of four out of five rows is one meter, while a 

row was planted at 0.7 meters.

Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) 

No preparation or maintenance works were done 

before and after planting the newly established 

culture (e.g. fertilization, irrigation, etc). 


The monitoring of the growth and development 

of the two willow species used to set up the 

experimental culture on the phosphogypsum waste 

pile, namely S. alba and S. fragilis, led to surprising 

results. Thus, on April 8, 2010, twelve days after the 

planting, it was observed that 107 out of the 110 

planted cuttings took root and sprouts had developed 

on them, while the first two-three leaves had already 

emerged in a small number of these cuttings. Three of 

the 110 cuttings did not take root and dried out. Two 

of these belong to S. fragilis and one to S. alba. On 

April 18, 2010, the sprouts on the 107 cuttings 

opened and the first leaves emerged. Some of them 

even grew three-four cm long shoots. On May 20, 

2010, it was observed that of the 107 cuttings that 

bore sprouts and shoots only 81 developed normally, 

with 5-20 cm long shoots. The other 26 were 

stagnating. The same situation was encountered on 

June 1, 2010, with the specification that the 81 

cuttings with normal development had 10-35 cm long 

shoots and some of the 26 stagnating cuttings began 

to dry. 

We must mention that on the two rows (one with 

ditch for the verification of the microclimate 

influence and one without ditch, as witness), where 

the planting depth was 0.7 meters, only S. fragilis was 

used. These two rows registered the highest number 

of stagnating cuttings about to get dry. The 

conclusion is thus that the planting depth is very 

important, the greater the depth, the more chances the 

cuttings have to take root and develop normally. By 

taking phosphogypsum samples from various depths 

and performing humidity analyses, it was observed 

that humidity increases directly proportionally with 

the depth of the sample. Moreover, by analyzing 

samples from the surface of the phosphogypsum (0-5 

cm) and those from the bottom of the ditches (40 cm), 

it was noticed over a period of several months that 

the samples from greater depths contained more 

water even though the months when the sample was 

collected were poor in precipitations. We specify that 

187 

no samples for the humidity test were collected in 

December 2009 because it rained on the day 

scheduled for the sample collection (December 12, 

2009). The graph in Fig. 2 presents the variation of 

humidity depending on the time and depth for the 

sample collection. 

Fig. 2. The variation of humidity depending on the 

time and depth of the sample collection 

This situation explains to a large extent the 

surprising presence of certain xerophyte species 

alongside hygrophyte ones on the phosphogypsum 

waste pile. 

In order to make a correct estimation of the 

rooting and normal development of the cuttings 

depending on species, we will only take into account 

the eight rows on which the cuttings were planted at a 

depth of one meter (four with ditch for the 

verification of the microclimate effect and four 

without ditch, as witness). These eight rows include 

four rows planted with S. alba and four rows planted 

with S. fragilis. The total number of cuttings on these 

eight rows is 88, of which S. alba – 44, and S. fragilis 

– 44. Of the 44 S. alba cuttings planted on four of the 

eight rows, 38 develop normally, while of the 44 S. 

fragilis cuttings planted on four of the eight 

rows, only 32 develop normally. Though it is 

premature to draw pertinent conclusions, we can say 

that S. alba seems to be better adapted to the 

conditions of the phosphogypsum waste pile, 

considering that its percentage of rooting and 

development is 86.36%, compared to S. fragilis 

whose percentage is 72.72%. Taking these results


into account, it is surprising why S. alba did not 

emerge spontaneously on the phosphogypsum waste 

pile, considering that we identified a female specimen 

from this species located at less than one km from the 

pile. This observation represents another argument in 

favor of the hypothesis that the species 

S. fragilis and S. matsudana existing on the 

phosphogypsum waste pile occurred by vegetative 

reproduction and not sexual one (from seeds). 

In regards to the lower rooting and development 

degree, it is probable that the age of the cuttings used 

for planting had an important role in this aspect. 

Thus, all the S. alba cuttings were young (under one 

year old), while those of S. fragilis were older 

(between two and three years old). 

The phosphogypsum on waste pile no. 3, 

belonging to the S.C. Fertilchim – Marway S.A. 

company, contains 90% calcium sulfate or gypsum 

hydrated with water molecules (CaSO4x2H2O), 

alongside which we can encounter phosphorus 

pentoxide (P2O5), traces of fluorhidric acid (HF), 

silicate (SiO2) and high concentrations of heavy 

metals [5]. An analysis bulleting released by the 

Constanta County Office for Agronomical Studies 

and Pedology on May 6, 2009 attests to the fact that 

the analyzed phosphogypsum contains no organic 

matter (humus), nor nitrogen (N). The nutrients 

contained by the phosphogypsum are potassium (K) 

in very low quantity and a higher amount of 

phosphorus (P), a fact also demonstrated by the 

analyses accomplished by the method of extraction 

with lactate acetate (A.L.), which were realized in the 

Pedology Laboratory of the Faculty for Natural and 

Agricultural Sciences within “Ovidius” University. 

Even though it is hard to believe that there are species 

that can develop normally on a 100% mineral 

substrate, these four willow species (S. fragilis, S. 

matsudana and S caprea – spontaneous, and S. alba 

– introduced artificially) contradict this statement. 

Another factor that makes possible the normal 

development of these species directly on 

phosphogypsum is their resistance in conditions of 

high soil salinity. Thus, S. fragilis, S. matsudana and 

S. seringeana tolerate high salinity values [6], while 

S. alba is “the most tolerant of all willow species to 

brackish water” [7]. In parallel, pH analyses were 

accomplished on samples collected from depths 

between 5 and 100 cm which displayed pH values 

188 

between 4.7 and 6.56. These results, corroborated 

with the fact that the quoted species prefer a slightly 

acid pH, make the phosphogypsum waste pile a 

favorable environment for the setting up of a willow 

culture. 

As the graph in Fig. 3 shows, no correlation can 

be made between pH value and the depth of sample 

collection. 

Fig. 3. The pH variation depending on the time and 

depth of the sample collection 

The only plausible explanation regarding this 

random distribution of the pH values in the 

phosphogypsum deposit could be that at the moment 

when the phosphogypsum suspension was 

neutralized, the milk of lime used did not always have 

the proper concentration. 

Willow is one of the well studied plants in order 

to use it in the phytoreparation processes, as it has a 

high capacity to accumulate heavy metals and it is 

easy to cultivate (Tremela et al. 1997; Pulford and 

Watson 2003) quoted by [8]. By concentrating 

important quantities of heavy metals in the shoots that 

will be collected every year, the willows will 

accomplish a depollution of the phosphogypsum and 

this will be a first step towards its transformation into 

organic-mineral fertilizer when the willow plantation 

will be eliminated. The strong bioaccumulation 

phenomenon in the species of the genus Salix will be 

favored by the slightly acid pH and will accelerate the 

cleansing of the phosphogypsum [9].

Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010) 

The harvesting should be done between 

November-February, after the leaves fall from the 

shoots. For harvesting, Claas Jaguar 880 GBE 1 or 

Claas Jaguar combines fitted with a HS2 harvesting 

head will be used. This type of combines transform 

the harvested shoots into a hash [10] that is left to dry 

and is then used in thermal power stations especially 

adapted for this solid fuel. The hash can be 

transformed into pellets used in regular thermal 

power stations as solid fuel. 

It is premature to speak about the role of the 

microclimate. By observing the phenotypical 

development of the willows planted in ditches and by 

comparing them to the willows planted directly on 

phosphogypsum, no major differences were noticed 

in regard to the length of the shoots and the plant 

vigor. In the case of the number of cuttings that 

display normal development, there are however small 

differences. Thus, in the case of S. fragilis, on the 

rows planted in ditches, there is a number of 17 

cuttings that develop normally, compared to only 15 

cuttings with normal development that were planted 

directly on phosphogypsum (witness area). In the 

case of S. alba on the rows planted in ditches, 20 

cuttings develop normally, compared to only 17 

cuttings with normal development planted directly on 

the phosphogypsum (witness area). It was noticed 

that the microclimate effect created by the ditches 

into phosphogypsum have a very important role in the 

case of seed germination and development of the 

annual herbaceous species. Thus, a few months after 

the digging of the ditches, a large number of 

herbaceous plants developed on their bottom. These 

plants germinated from seeds brought by the wind, 

mostly belonging to the dominant species in the waste 

pile phytocoenosis, Puccinellia distans. 


In order to set up a willow culture, it is best to 

harvest and plant the cuttings between February 15 – 

March 15. 

The planting depth is very important. It was 

observed that at depths exceeding 40 cm, 

phosphogypsum always displays humidity over 25% 

even if the sample was collected after a period with 

189 

no precipitations. The optimum planting depth is 100 

cm. 

The age of the cuttings has a very important role 

in the rooting process and their normal development. 

Thus, in the case of the one-year-old cuttings, the 

success percentage was higher. 

Over the entire surface of the waste pile, between 

0 and 100 cm, the distribution of the pH values is 

purely random and they range between a minimum of 

4.7 and a maximum of 6.56. This fact 

favors the development of species of the genus Salix, 

which prefer a substrate with slightly acid pH. 

The total lack of organic matter and of nitrogen 

from the substrate does not prevent the species from 

the genus Salix to develop normally, but it is possible 

to lead to a lower quantity of wood mass per surface 

unit. 

Even though within the experimental culture 

there was a larger number of cuttings of the species S. 

alba with normal development, it is premature to say 

that this species is better adapted to the 

environmental conditions than S. fragilis, a 

spontaneous species in the phosphogypsum waste pile 

ecosystem. 

It is also premature to draw a conclusion about 

the influence of the microclimate generated by the 

ditches into phosphogypsum on the development of 

the S. alba and S. fragilis cuttings. However, it was 

noticed that the microclimate generated by the ditches 

has a beneficial influence on the species of annual 

plants. Thus, in an interval of three months, a large 

number of herbaceous plants emerged on the bottom 

of the ditches, most of them belonging to Puccinellia 

distans, a dominant species in the phytocoenosis of 

the phosphogypsum deposit. 

The advantages of a culture with species 

belonging to the genus Salix on the phosphogypsum 

waste piles are multiple: 

- To obtain ecological fuel – the carbon 

eliminated by burning represents the carbon 

- Stored previously by photosynthesis, so no extra 

amounts of carbon are released into the 

atmosphere; 

- To use fields otherwise improper for other 

cultures and transform thus losses into profit; 

- The development of the root system and of the 

willow shoots will prevent wind erosion;


- Taking into account the fact that the harvest of 

shoots takes place between November and 

February, the annual leaf litter on the 

phosphogypsum surface will accelerate the 

process of soil formation; 

- By the phenomenon of bioaccumulation, the 

trees (shrubs) from the plantation will 

concentrate into their own structures important 

quantities of heavy metals that will be removed 

annually by cutting the shoots and decreasing 

thus the polluting content of the 

phosphogypsum; 

- The species of the genus Salix, fond of 

humidity, will retain part of the water resulted 

from precipitations, reducing drastically the 

levigation phenomenon and the draining of the 

phosphorus into the ground water; 

- The photosynthesis and evapo-transpiration 

generated by the willows will improve air 

quality and the local microclimate during the 

warm season. 

The main disadvantage is the fact that, being a 

monoculture, it will be more vulnerable to pests. 

Another possible disadvantage is that, because the 

cuttings are not planted like in a culture on swampy 

or irrigated land (the same density per square meter), 

the quantity of wood mass per ha can be reduced. 

5. References 

[1]. SÂRBU I., Stefan N., Ivănescu Lăcrămioara, 

Mânzu C., 2001. Flora ilustrată a plantelor 

vasculare din estul României, Determinator, vol. 

I şi II, Editura Universităţii „Alexandru Ioan 

Cuza”, Iaşi. 

[2]. GOMOIU M.-T., Skolka M., 2001. Ecologie - 

Metodologii pentru studii ecologice, Ovidius 

University Press, Constanţa. 

[3]. SKVORTSOVA. K., 1999. Willows of Russia 

and adjacent countries. Taxonomical and 

geographical review. Univ. Joensuu Fac. 

Mathem. and Natru. Sci. Rept. Ser. 39. 307 pp. 

[4]. www.bfafh.de/inst2/sg-pdf/52_3-4_148.pdf. 

Diversity of dte willow complex Salix alba – S x. 

rubens – S. Fragilis 

190 

[5]. www.containment.fsu.edu/cd/content/pdf/466.pd 

f. Vegetative cover for phosphogypsum dumps: 

A Romanian field study. 

[6]. CROUCH R.J, Honeyman M.N., 1986, 'The 

relative salt tolerance of willow cuttings.' Journal 

of Soil Conservation, vol 42 (2), p. 103-104. 

[7]. ZALLAR S. Botanical Characteristics of the 

Willows, Soil Conservation Authority, Kew. 

[8]. www.sci.uszeged.hu/ABS/2006/Acta%20HP/50 

37.pdf. Change of root and rhizosphere characters 

of willow (Salix sp) induced by high heavy metal 

pollution. 

[9]. www.umass.edu/.../Phytoremediation%20PDF 

/PhytoLitReview.pdf. Phytoremediation literature 

review. 

[10]. www.bioeng.ca/pdfs/meetingpapers/2005/CSAE%20papers/05-080.pdf. 

Cutting, bundling and chipping shortrotation 

willow.

VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

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