VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole
VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole
VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole
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An<strong>ale</strong>le Universităţii Ovidius<br />
Seria: BIOLOGIE – ECOLOGIE<br />
Volumul 14, anul 2010<br />
<strong>VOLUM</strong> <strong>OMAGIAL</strong><br />
Ovidius University Annals<br />
BIOLOGY – ECOLOGY Series<br />
Volume 14, year 2010<br />
OVIDIUS UNIVERSITY PRESS
An<strong>ale</strong>le Universităţii Ovidius, Seria Biologie – Ecologie<br />
Volumul 14 (2010)<br />
<strong>VOLUM</strong> <strong>OMAGIAL</strong><br />
(<strong>de</strong>dicat împlinirii a 20 <strong>de</strong> ani <strong>de</strong> la înfiinţarea Facultăţii<br />
<strong>de</strong> <strong>Ştiinţe</strong> <strong>ale</strong> <strong>Naturii</strong> <strong>şi</strong> <strong>Ştiinţe</strong> <strong>Agricole</strong>)<br />
Redactor Şef<br />
Prof. univ. dr. Marian Traian GOMOIU<br />
Membru corespon<strong>de</strong>nt al Aca<strong>de</strong>miei Române<br />
mtg@datanet.ro<br />
Redactori<br />
Conf. univ. dr. Marius FĂGĂRAŞ Prof. univ. dr. Rodica BERCU<br />
fagaras_marius@yahoo.com rodicabercu@yahoo.com<br />
Mail address: Faculty of Natural and Agricultural Sciences, “Ovidius” University of Constanţa, Aleea<br />
Universităţii nr. 1, corp B, Constanţa RO-900470, România, Tel. 0241605060/ Fax: 0241606432,<br />
contact@stiintele-naturii.ro.<br />
ORDERING INFORMATION<br />
Ovidius University Annals of Biology – Ecology is published annually by Ovidius University Press. The journal<br />
may be obtained on exchange basis with similar Romanian or foreign institutions.<br />
No part of its publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any<br />
means, electronically, mechanical, photocopying, recording or otherwise, without the written permission of the<br />
Publisher, Ovidius University Press, Blvd. Mamaia 124, RO-900527, Constanţa, România.<br />
© 2010 Ovidius University Press ISSN 1453–1267
Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010)<br />
Contents<br />
Limitative mycotic factors for some plants from the Bulgarian coast of the Black Sea<br />
Gavril NEGREAN………………………………………………………………………................................<br />
The medicinal plants of Provadiisko Plateau<br />
Dimcho ZAHARIEV, Desislav DIMITROV………………………………………………………………...<br />
The plants with protection statute, en<strong>de</strong>mites and relicts of the Shumensko Plateau<br />
Dimcho ZAHARIEV, Elka RADOSLAVOVA……………………………………………………………...<br />
A characteristic of mo<strong>de</strong>l habitats in south Dobrudja<br />
Dimcho ZAHARIEV………………………………………………………………………..………………..<br />
Floristic aspects of the Hills of Camena village (Tulcea county)<br />
Marius FĂGĂRAŞ............................................................................................................................................<br />
I<strong>de</strong>ntification of some rose genitors with resistance to the pathogens agents attack<br />
Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU, Elena DOROFTEI ………...<br />
Preliminary data on Meledic-Mânzăleşti Natural Reserve (Buzău county, Romania)<br />
Daciana SAVA, Mariana ARCUŞ, Elena DOROFTEI………………………………………………………<br />
Contributions to the biometrical and phytobiological study on wild garlic<br />
Mariana LUPOAE, Dragomir COPREAN, Rodica DINICĂ, Paul LUPOAE……………………………….<br />
Dinitrophenyl <strong>de</strong>rivates action on wheat germination<br />
Cristina Amalia DUMITRAŞ -HUŢANU ………………………….………………………………………..<br />
The action of some insectici<strong>de</strong>s upon physiological indices in Rana (Pelophylax) ridibunda<br />
Alina PĂUNESCU, Cristina M. PONEPAL, Octavian DRĂGHICI, Alexandru G. MARINESCU..............<br />
Changes of some physiological parameters in Prussian carp un<strong>de</strong>r the action of some fungici<strong>de</strong><br />
Maria C. PONEPAL, Alina PĂUNESCU, Alexandru G. MARINESCU, Octavian DRĂGHICI...................<br />
Cytogenetic effects induced by manganese and lead microelements on germination at Triticum<br />
aestivum L.<br />
Elena DOROFTEI, Maria Mihaela ANTOFIE, Daciana SAVA, Marioara TRANDAFIRESCU...................<br />
Problems of the harmonizing environmental legislation at the compartment “Pisces” in<br />
the Republic of Moldova<br />
Petru COCIRTA, Olesea GLIGA …………………………………………………………………………....<br />
Biodiversity conservation in Constanţa county<br />
Silvia TURCU, Marcela POPOVICI, Loreley JIANU.....................................................................................<br />
ISSN-1453-1267 © 2010 Ovidius University Press<br />
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Ovidius University Annals of Natural Sciences, Biology – Ecology Series, Volume 14 (2010)<br />
The present situation of the nose horned viper populations (Vipera ammodytes montandoni<br />
Boulenger 1904) from Dobrudja (Romania and Bulgaria)<br />
Marian TUDOR………………………………………………………………………………………………<br />
Body size variation in Rana temporaria populations inhabiting extreme environments<br />
Rodica PLĂIAŞU, Raluca BĂNCILĂ, Dan COGĂLNICEANU……………………………………………<br />
Utilization of epifluorescence microscopy and digital image analysis to study some morphological<br />
and functional aspects of prokariotes<br />
Simona GHIŢĂ, Iris SARCHIZIAN, Ioan ARDELEAN…………………………………………………….<br />
Changes in bacterial abundance and biomass in sandy sediment microcosm supplemented with<br />
gasoline<br />
Dan Răzvan POPOVICIU, Ioan ARDELEAN.................................................................................................<br />
The formation of bacterial biofilms on the hydrophile surface of glass in laboratory static<br />
conditions: the effect of temperature and salinity<br />
Aurelia Manuela MOLDOVEANU, Ioan I. ARDELEAN...............................................................................<br />
The clinical utility of additional methods in effusions evaluation<br />
Ana Maria CREŢU, Mariana AŞCHIE, Diana BADIU, Natalia ROŞOIU…………………………………..<br />
Spatio-temporal dynamics of phytoplankton composition and abundance from the Romanian Black<br />
Sea coast<br />
Laura BOICENCO……………………………………………………………………………………………<br />
Aspects regarding the biodiversity of the aquatic and semi-aquatic heteroptera in the lakes situated<br />
in the middle basin of the Olt River<br />
Daniela Minodora ILIE.....................................................................................................................................<br />
Program of prevention and control of fungus infestation of grain and fod<strong>de</strong>r, human and animal<br />
protection against mycotoxins<br />
Ioan Aurel POP, Augustin CURTICĂPEAN, Alin GULEA, Cornel PODAR, Iustina LOBONTIU..............<br />
Data on the dynamics of some microbial groups in soils with different trophic status in Cumpăna<br />
region (Dobrudja)<br />
Elena DELCĂ………………………………………………………………………………………………...<br />
The agricultural potential of phosphogypsum waste piles<br />
Lucian MATEI………………………………………………………………………………………………..<br />
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Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
LIMITATIVE MYCOTIC FACTORS FOR SOME PLANTS FROM THE<br />
BULGARIAN COAST OF THE BLACK SEA<br />
Gavril NEGREAN<br />
Universitatea din Bucureşti, Grădina Botanică, Şoseaua Cotroceni nr. 32, Bucureşti<br />
__________________________________________________________________________________________<br />
Abstract: We present a list of 119 parasitic fungi collected in Bulgaria from the following groups:<br />
Peronospor<strong>ale</strong>s, Ascomycetes, Uredin<strong>ale</strong>s, Ustilagin<strong>ale</strong>s, Agaric<strong>ale</strong>s, Polypor<strong>ale</strong>s, Gasteromycet<strong>ale</strong>s and Fungi<br />
Anamorphici. An alien rust new for Bulgaria is also found (Puccinia komarovii).<br />
There are also some commentaries regarding the rare plants guested by different fungi; other fungi may<br />
contribute to the diminishing of the damages produces by some weeds; we draw attention about some foreign<br />
fungi with invasive character.<br />
Keywords: New parasitic fungi for Bulgaria, invasive fungi, matrix nova, Dobrogea, Bulgaria.<br />
__________________________________________________________________________________________<br />
1. Introduction.<br />
Following our preoccupa-tion regarding the<br />
limitative factors for the vascular plants on the<br />
Black Sea, we present the results of our<br />
investigations on the Bulgarian Dobrogean Black<br />
Sea si<strong>de</strong>. Our observations from the previous years<br />
were published within several notes [1, 2, 3, 4, 19].<br />
2. Material and Methods.<br />
The fungi were collected from the areas nearby<br />
the sea si<strong>de</strong> between Duranculac and the<br />
embouchure of the Batovo valley, in April and June<br />
2006 and April – October 2008. A very small<br />
amount of fungi collectetd from other areas of<br />
Bulgaria, they also listed. The big majority are<br />
coming from the Dobrich district. The materials<br />
were collected on the way and their conditioning<br />
was donje in conformity with the usual techniques<br />
and <strong>de</strong>terminated by help of the instruments we had<br />
at our disposal [5, 6, 7, 8, 9, 10, 11, 12, 13, 14].<br />
The nomenclature of the authors of the hosts<br />
after Flora Romaniae [15] and Flora Europaea [16,<br />
17]. The conditionated and <strong>de</strong>termined materials<br />
were <strong>de</strong>posited in the Herbarium of the University<br />
from Bucureşti [BUC] and partially in the<br />
Herbarium of the Botanic Institute from Sofija<br />
[SOM]. The list is alphabetically coordinated, on<br />
big groups offungi and the coronims from North to<br />
South.<br />
3. Results and Discussions<br />
In these two years mentioned, there were<br />
collected 217 specimens, representing the analized<br />
groups of fungi (Table 1). Apparently, a number of<br />
16 combinations fungus – host plant („matrix<br />
nova”), incase of the Peronospor<strong>ale</strong>s (Table 2), and<br />
were not indicated since Bulgaria [14]. Among<br />
Erysiphaceae, 19 combinations [8], alike species<br />
have not been found in Bulgaria. Likewise, a<br />
number of 15 combinations between Uredin<strong>ale</strong>s [7]<br />
do not seem to be cited from Bulgaria. Puccinia<br />
komarovii rust, guesting the alien plant Impatiens<br />
parviflora DC. is new for the Bulgarian mycobiota.<br />
Sozological aspects.<br />
Following a long cohabitation (co evolution)<br />
between fungi and their hosts there has been created<br />
an equilibrum, so that we have barely noticed<br />
ruptures of this equilibrum. Among the rare guested<br />
plants, we mention the following: Astragalus<br />
cornutus (important damages locally), Buglossoi<strong>de</strong>s<br />
arvensis subsp. sibthorpiana, Centaurea<br />
salonitana, Centaurea thracica, Clypeo-la<br />
jonthlaspi, Dianthus leptopetalus, Euphorbia<br />
myrsinites, Gypsophila pallasii, Hieracium bauhinii,<br />
Leymus racemosus subsp. sabulosus, Limonium<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
meyeri, Onosma rigidum, Potentilla taurica, Rhagadiolus<br />
stellatus, Scilla bithynica, Sherardia arvensis<br />
L. subsp. maritima etc.<br />
Some plants are extremely important from the<br />
sozological point of view, taking into consi<strong>de</strong>ration<br />
that they are in contact with new climatic<br />
conditions being subjected to the eventual<br />
speciation phenomena. It will be the case of Astragalus<br />
cornutus, Beta trigyna, Carduus pycnocephalus,<br />
Euphorbia dobrogensis, Medicago arabica,<br />
Pimpinella peregrina, Plumbago europaea, Ranunculus<br />
oxyspermus, Rumex tuberosus subsp. tuberosus,<br />
Scilla bithynica etc.<br />
Some fungi can have a certain play in<br />
diminishing the population of some weeds,<br />
contributiong this way to the diminution of the<br />
damages they produce, such as Amaranthus retroflexus,<br />
Avena fatua, Bassia scoparia, Carduus<br />
acanthoi<strong>de</strong>s, Lycium barbarum, Malva sylvestris,<br />
Picris echioi<strong>de</strong>s (carantin plant), Rumex patientia<br />
etc.<br />
I collecting some alien fungi with invasiv<br />
character in present (Puccinia malvacearum, Erysiphe<br />
mougeotii, Puccinia helianthi) or in future<br />
(Puccinia komarovii, Puccinia pelargonii-zonalis).<br />
The hyperparasite Ampelomyces quisqua-lis,<br />
also contributes the diminution of the damnages<br />
produced by some mil<strong>de</strong>w, there have been registered<br />
some cases.<br />
Last but not least, we consi<strong>de</strong>r that the fungi<br />
have their right to live.<br />
4. Conclusions.<br />
From the 115 fungi collec-ted from the seasi<strong>de</strong><br />
of the Bulgarian Dobrogea, the most majority are<br />
plants parasites. Most of them belong the groups:<br />
Peronspor<strong>ale</strong>s, Erysiphaceae, Uredin<strong>ale</strong>s and Fungi<br />
Anamorphici. The results are important ones: a new<br />
adventitious parasite rust for the Bulgarian<br />
mycobiota and a number of 50 combinations from<br />
Bulgaria apparently not o<strong>de</strong>ntiofied in their form by<br />
now. We ascertained that following a long coexistence,<br />
between fungi and their hosts, the plants,<br />
there has been created a rather stable equilibrium.<br />
4<br />
LIST OF SPECIES<br />
PERONOSPORALES<br />
Albugo amaranthi (Schwein.) O. Kuntze<br />
(Wilsonia bliti (Biv.) Thines), matrix:<br />
Amaranthus retroflexus L. - Camen Briag,<br />
centrum, in locis ru<strong>de</strong>ralis, 43º27′20.83″N,<br />
28º33′04.63″E, alt. circa 35m, 23 X 2008, G.<br />
Negrean (11.594) [BUC]. Cavarna S, prope hotel,<br />
in locis ru<strong>de</strong>ralis, 43º21′17.41″N, 28º21′17.41″E,<br />
alt. circa 30 m, 10 VIII 2008, G. Negrean (11.484)<br />
[BUC].<br />
Albugo candida (Pers.) Roussel, matrix:<br />
Alyssum <strong>de</strong>sertorum Stapf - Bălgarevo E, Cap<br />
Caliacra W 2 km, in herbosis et petrosis, 14 IV<br />
2006, G. Negrean (7065c) [BUC].<br />
Alyssum hirsutum Bieb. - Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, G.<br />
Negrean (10.220) [BUC].<br />
Camelina rumelica Velen. - Cavarna E, in<br />
herbosis, 12 IV 2008, G. Negrean (10.256) [BUC].<br />
Capsella bursa-pastoris (L.) Medicus - Balcic,<br />
centrum, in cortis moscheii, ru<strong>de</strong>ral, 13 IV 2006, G.<br />
Negrean (7043) [BUC]. Balcic, centrum, ru<strong>de</strong>ral, 4<br />
VI 2006, G. Negrean (7252) [BUC]. Sofija S, prope<br />
Hotel Vitosha (N), ru<strong>de</strong>ral, 24 VI 2006, G. Negrean<br />
(7370).<br />
Clypeola jonthlaspi L. - Bălgarevo E, ut Cap<br />
Caliacra, in herbosis, 14 IV 2006, G. Negrean<br />
(7072a) [BUC]. Cavarna E, in herbosis,<br />
43º24′25.14″N, 28º22′19.22″E, alt. circa 60 m, 12<br />
IV 2008, G. Negrean (10.291) [BUC].<br />
Sisymbrium loeselii L. - Sofija S, prope Univ.<br />
Technica, 21 VI 2006, G. Negrean (7323) [BUC].<br />
Sofija S, prope Hotel Vitosha (N), ru<strong>de</strong>ral, 24 VI<br />
2006, G. Negrean (7382) [BUC].<br />
Sisymbrium orient<strong>ale</strong> L. s. l. - Duranculac E, ad<br />
littore Mare Nigrum, in locis ru<strong>de</strong>ralis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V<br />
2008, G. Negrean (10.436) [BUC].<br />
Albugo portulacae (DC. ex Duby) O. Kuntze,<br />
matrix:<br />
Portulaca oleracea L. subsp. oleracea - Sozopol,<br />
in arenosis, 42º24′05.22″N, 27º42′33.32″E, alt.<br />
circa 15 m, 9 VIII 2008, G. Negrean (11.888)<br />
[BUC].
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Albugo tragopogonis (DC.) Gray, matrix:<br />
Xeranthemum annuum L. - Cavarna E, in<br />
herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa<br />
60 m, 12 IV 2008, G. Negrean (11.504) [BUC].<br />
Ecrene N, ad oram rivuli Batova, in arenosis,<br />
43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 13 IV<br />
2006, G. Negrean (7050) [BUC].<br />
Bremia lactucae Regel, matrix:<br />
Carduus acanthoi<strong>de</strong>s L. - Bălgarevo E, Cap<br />
Caliacra, vers E, ut Mare Nigrum, in herbosis<br />
ru<strong>de</strong>ralis, 15 IV 2006, G. Negrean (7074a) [BUC].<br />
Cavarna SW, Bojorets S, Caliacria, in locis<br />
ru<strong>de</strong>ralis, 43º25′03.47″N, 28º16′48.25″E, alt. circa<br />
46 m, 22 X 2008, G. Negrean (11.521) [BUC].<br />
Picris echioi<strong>de</strong>s L. - Camen Briag, in locis<br />
ru<strong>de</strong>ralis, 43º27′18.92″N, 28º33′14.19″E, alt. circa<br />
35 m, 23 X 2008, G. Negrean (11.551) [BUC].<br />
Rhagadiolus stellatus (L.) Gaertn. - Rusalca, sub<br />
platous prope littore Mare Nigrum, in abruptis et<br />
silvis, 43º24′733″N, 28º29′780″E, alt. circa 60 m,<br />
10 V 2008, G. Negrean (10.459) [BUC].<br />
Crepis pulchra L. - Bălgarevo E, Cap Caliacra N,<br />
sinistra vallis Bolata-Dere, in herbosis,<br />
43º22′59.37″N, 28º28′18.08″E, alt. circa 2 m, 4 VI<br />
2006, G. Negrean (7397) [BUC].<br />
Hyaloperonospora parasitica (Pers.: Fr.)<br />
Constant., matrix:<br />
Alyssum <strong>de</strong>sertorum Stapf - Bălgarevo E, Cap<br />
Caliacra W 2 km, in herbosis et petrosis, 14 IV<br />
2006, G. Negrean (7065b) [BUC].<br />
Hyaloperonospora tribulina (Pass.) Constant.<br />
(Peronospora tribulina Pass.), matrix:<br />
Tribulus terrestris L. - Cavarna SW, Caliacria, in<br />
locis ru<strong>de</strong>ralis, 43º25′03.47″N, 28º16′48.25″E, alt.<br />
circa 46 m, 22 X 2008, G. Negrean (11.511)<br />
[BUC].<br />
Peronospora aestivalis H. Syd., matrix:<br />
Melilotus sp. - Balcic W, in herbosis, 3 VI 2006,<br />
G. Negrean (7225) [BUC].<br />
Peronospora alsinearum Casp., matrix:<br />
Stellaria media (L.) Vill. s. l., Ecrene N, ad oram<br />
rivuli Batova, 43º21′10.43″N, 28º04′31.74″E, alt.<br />
circa 2 m, 13 IV 2006, G. Negrean (7820) [BUC].<br />
Peronospora alta Fuckel, matrix:<br />
Plantago major L. subsp. major, Sofija S, prope<br />
Hotel Moskva, 22 VI 2006, G. Negrean (7328)<br />
[BUC].<br />
Peronospora arborescens (Berk.) <strong>de</strong> Bary,<br />
matrix:<br />
5<br />
Papaver dubium L. - Bălgarevo E, Cap Caliacra,<br />
vers E, ut Mare Nigrum, in herbosis ru<strong>de</strong>ralis,<br />
43º22′03.97″N, 28º27′57.08″E, alt. circa 25 m, 15<br />
IV 2006, G. Negrean (7079) [BUC].<br />
Peronospora astragalina Syd., matrix:<br />
Astragalus hamosus L. - Crapetz E, prope Cap<br />
Crapetz, solo arenoso, 43º38′23.64″N,<br />
28º34′25.86″E, alt. circa 6 m, 12 IV 2008, G.<br />
Negrean (10.242) [BUC].<br />
Peronospora calotheca <strong>de</strong> Bary, matrix:<br />
Galium aparine L. - Crapetz E, prope Cap Crapetz,<br />
solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt. circa<br />
6 m, 12 IV 2008, G. Negrean (10.246) [BUC].<br />
Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo<br />
terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa 15<br />
m, 12 IV 2008, G. Negrean (10.270) [BUC].<br />
Peronospora conglomerata Fuckel, matrix:<br />
Erodium ciconium (L.) L’Herit. - Cavarna S, in<br />
arenosis, sub Collina Cheracman, 12 IV 2008, G.<br />
Negrean (10.241) [BUC]. Cavarna SW, Bojorets S,<br />
Caliacria, in locis ru<strong>de</strong>ralis, 43º25′03.47″N,<br />
28º16′48.25″E, alt. circa 46 m, 22 X 2008, G.<br />
Negrean (11.588) [BUC].<br />
Peronospora farinosa (Fr.) Fr., matrix:<br />
Bassia scoparia (L.) A. J. Scott, Sofija S, cartier<br />
S, ru<strong>de</strong>ral, 22 VI 2006, G. Negrean (7330) [BUC].<br />
Chenopodium album (Boiss.) Kuntze - Camen<br />
Briag, Motel, in locis ru<strong>de</strong>ralis, 43º27′13.83″N,<br />
28º33′04.96″E, alt. circa 25 m, 7 VI 2008, G.<br />
Negrean (10.707) [BUC].<br />
Chenopodium opulifolium Schrad. ex Koch & Ziz<br />
- Cavarna S, prope hotel, in locis ru<strong>de</strong>ralis,<br />
43º21′17.41″N, 28º21′17.41″E, alt. circa 30 m, 10<br />
VIII 2008, G. Negrean (11.484) [BUC].<br />
Peronospora ficariae L.R. Tul. ex <strong>de</strong> Bary,<br />
matrix:<br />
Ranunculus ficaria L. subsp. calthifolius<br />
(Reichenb.) Arcangeli - Balcic W, in locis umbrosis,<br />
15 IV 2006, G. Negrean (7090) [BUC].<br />
Peronospora medicaginis-minimae<br />
Gaponenko, matrix:<br />
Medicago lupulina L., Sofija S, prope Hotel<br />
Vitosha, ru<strong>de</strong>ral, 22 VI 2006, G. Negrean (7321)<br />
[BUC].<br />
Peronospora sherardiae Fuckel, matrix:<br />
Sherardia arvensis L. subsp. maritima (Griseb.)<br />
Soják - Bălgarevo E, Cap Caliacra, in herbosis,<br />
43º22′03.97″N, 28º26′57.08″E, alt. circa 25 m, 12
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
IV 2008, G. Negrean (10.234) [BUC]. Balcic W, in<br />
herbosis, 15 IV 2006, G. Negrean (7087a) [BUC].<br />
Peronospora tribulina Pass. = Hyaloperonospora<br />
tribulina (Pass.) Constant.<br />
Peronospora v<strong>ale</strong>rianellae Fuckel, matrix:<br />
V<strong>ale</strong>rianella sp. - Crapetz E, prope Cap Crapetz,<br />
solo arenoso, 43º38′23.64″N, 28º34′25.86″E, alt.<br />
circa 6 m, 12 IV 2008, G. Negrean (10.245) [BUC].<br />
Peronospora viciae (Berk.) <strong>de</strong> Bary, matrix:<br />
Vicia sativa L. subsp. nigra (L.) Ehrh. -<br />
Duranculac S, in herbosis, 43º39′53.06″N,<br />
28º31′15.26″E, alt. c. 12 m, 13 IV 2006, G.<br />
Negrean (7036) [BUC]. Rusalca NNE, in herbosis,<br />
43º25′34.94″N, 28º31′51.46″E, alt. circa 8 m, 12 IV<br />
2008, G. Negrean (10.277) [BUC].<br />
Plasmopara nivea (Unger) J. Schröt.<br />
(Plasmopara umbelliferarum (Casp.) J. Schröt. ex<br />
Wartenw.), matrix:<br />
Aegopodium podagraria L., Sofija S, Montes<br />
Vitosha, in herbosis subalpinis, 23 VI 2006, G.<br />
Negrean (7353) [BUC].<br />
ASCOMYCOTA<br />
Blumeria graminis (DC. ) Speer, matrix:<br />
Avena fatua L. - Rusalca, sub platou prope littore<br />
Mare Nigrum, in herbosis, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 7 VI 2008, G.<br />
Negrean (10.646) [BUC].<br />
Aegilops lorentii Hochst. - Rusalca, sub platou<br />
prope littore Mare Nigrum, in herbosis,<br />
43º24′750″N, 28º29′790″E, alt. circa 15 m, 10 V<br />
2008, G. Negrean (10.465) [BUC].<br />
Hor<strong>de</strong>um bulbosum L. Dobrogea, Shabla E, ad<br />
littore Mare Nigrum, in locis herbosis,<br />
43º33′707″N, 28º35′553″E, alt. circa 2 m, 9 V<br />
2008, G. Negrean (11.572) [BUC].<br />
Daldinia concentrica (Bolton) Ces. & De Not.<br />
- matrix: in lignos, Duranculac E, ad littore Mare<br />
Nigrum, in locis arenosis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, Leg. P.<br />
Anastasiu, <strong>de</strong>t. G. Negrean (11.593) [BUC].<br />
Epichloe typhina (Pers.: Fr.) Tul., matrix:<br />
Dactylis glomerata L. s. l., Sofija S, prope Hotel<br />
Moskva, in herbosis, 22 VI 2006, G. Negrean<br />
(7331).<br />
Erysiphe alphitoi<strong>de</strong>s (Griffon & Maubl.) U.<br />
Braun & S. Takam. (Microsphaera alphitoi<strong>de</strong>s<br />
Griffon & Maubl.), matrix:<br />
6<br />
Quercus pubescens Willd. - Bălgarevo E,<br />
Rusalca, prope littore Mare Nigrum, sub abruptum,<br />
10 VIII 2008, G. Negrean (11.503) [BUC].<br />
Quercus robur L., Sofija S, prope Hotel Vitosha<br />
(N), ru<strong>de</strong>ral, 24 VI 2006, G. Negrean (7379).<br />
Erysiphe aquilegiae DC., matrix:<br />
Aquilegia vulgaris L., - Camen Briag, centrum, in<br />
locis ru<strong>de</strong>ralis, subspont., 43º27′20.83″N,<br />
28º33′04.63″E, alt. circa 35 m, 23 X 2008, G.<br />
Negrean (11.536, T) [BUC; CL].<br />
Erysiphe artemisiae Grev., matrix:<br />
Artemisia vulgaris L. - Camen Briag, centrum, in<br />
locis ru<strong>de</strong>ralis, 43º27′20.83″N, 28º33′04.63″E, alt.<br />
circa 35 m, 23 X 2008, G. Negrean (11.532) [BUC].<br />
Erysiphe astragali DC., matrix:<br />
Astragalus hamosus L. - Rusalca, prope littore<br />
Mare Nigrum, in herbosis, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 7 VI 2008, G.<br />
Negrean (10.660) [BUC].<br />
Erysiphe buhrii U. Braun, matrix:<br />
Gypsophila pallasii Ikonn. - Bălgarevo E, <strong>de</strong>xtra<br />
vallis Bolata Dere, prope littore Mare Nigrum, in<br />
herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35<br />
m, 20 VII 2006, G. Negrean (11.450) [BUC].<br />
Erysipe cichoracearum DC., matrix:<br />
Centaurea salonitana Vis. - Rusalca N, ad littore<br />
Mare Nigrum, in herbosis, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 7 VI 2008, G.<br />
Negrean (10.720) [BUC]. Yailata, platou prope<br />
littore Mare Nigrum, in saxosis, 43º26′552″N,<br />
28º32′930″E, alt. circa 25 m, 8 VIII 2008, G.<br />
Negrean (11.472) [BUC]. Bălgarevo E, Cap<br />
Caliacra, in herbosis, 43º22′982″N, 28º26′4599″E,<br />
alt. circa 72 m, 8 VI 2008, G. Negrean (10.753)<br />
[BUC]. Balcic E, supra Tuzlata, in herbosis<br />
abruptis, 8 VI 2008, G. Negrean (10.747) [BUC].<br />
Crepis foetida L. subsp. rhoeadifolia (Bieb.)<br />
Čelak., Sofija S, prope Hotel Vitosha (N), ru<strong>de</strong>ral,<br />
24 VI 2006, G. Negrean (7375).<br />
Crepis pulchra L., Sofija S, prope Hotel Vitosha<br />
(N), ru<strong>de</strong>ral, 24 VI 2006, G. Negrean (7371).<br />
Lactuca viminea L. - Rusalca, prope littore Mare<br />
Nigrum, in locis herbosis et petrosis, 43º24′733″N,<br />
28º29′776″E, alt. circa 45 m, 10 V 2008, G.<br />
Negrean (10.454) [BUC].<br />
Tragopogon dubius Scop., Sofija S, prope Hotel<br />
Vitosha (N), ru<strong>de</strong>ral, 24 VI 2006, G. Negrean<br />
(7376).
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Erysiphe cruciferarum Opiz ex L. Junell,<br />
matrix:<br />
Alliaria petiolata (Bieb.) Cavara & Gran<strong>de</strong>,<br />
Sofija S, prope Hotel Moskva, ru<strong>de</strong>ral, 22 VI 2006,<br />
G. Negrean (7324).<br />
Alyssum hirsutum Bieb. - Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, G.<br />
Negrean (10.221) [BUC].<br />
Brassica nigra C. Koch - Bălgarevo SE, ad<br />
littore Mare Nigrum, sub abruptum, prope ferma<br />
pisciculturae, in locis herbosis, 43º25′02.83″N,<br />
28º31′00.18″E, alt. circa 5 m, 10 VIII 2008, G.<br />
Negrean (11.495) [BUC].<br />
Erysiphe cynoglossi (Wallr.) U. Braun, matrix:<br />
Buglossoi<strong>de</strong>s arvensis (L.) I. M. Johnston subsp.<br />
sibthorpiana (Griseb.) R. Fernan<strong>de</strong>s - Balcic W,<br />
Cap Caliacra, in herbosis, 3 VI 2006, G. Negrean<br />
(7295) [BUC].<br />
Echium italicum L. subsp. pyramidatum (DC.) . -<br />
Bălgarevo SE, Cap Caliacra, in herbosis,<br />
43º23′02.13″N, 28º26′53.26″E, alt. circa 30 m, 10<br />
VIII 2008, G. Negrean (11.480) [BUC].<br />
Echium vulgare L., Sofija S, prope Hotel Vitosha<br />
(N), ru<strong>de</strong>ral, 25 VI 2006, G. Negrean (7383)<br />
[BUC].<br />
Onosma rigidum Le<strong>de</strong>b. - Yailata, prope littore<br />
Mare Nigrum, in abruptis, 43º26′552″N,<br />
28º32′930″E, alt. circa 45 m, 7 VI 2008, G.<br />
Negrean (11.124, A) [BUC].<br />
Erysiphe <strong>de</strong>pressa (Wallr.) Schltdl – A,<br />
matrix:<br />
Onopordum acanthium L. - Cavarna SW, Bojorets<br />
S, Caliacria, 43º25′03.47″N, 28º16′48.25″E, alt.<br />
circa 46 m, 22 X 2008, G. Negrean (11.525) [BUC].<br />
Erysiphe g<strong>ale</strong>opsidis DC. = Neoerysiphe<br />
g<strong>ale</strong>opsidis (DC.) U. Braun<br />
Erysipe heraclei DC., matrix:<br />
Myrrhoi<strong>de</strong>s nodosa (L.) Cannon - Rusalca, sub<br />
platou prope littore Mare Nigrum, in herbosis,<br />
43º24′750″N, 28º29′790″E, alt. circa 15 m, 10 V<br />
2008, G. Negrean (10.465) [BUC].<br />
Scandix pecten-veneris L. subsp. pecten-veneris -<br />
Rusalca, sub platou prope littore Mare Nigrum, in<br />
herbosis, 43º25′116″N, 28º31′126″E, alt. circa 10<br />
m, 7 VI 2008, G. Negrean (10.650) [BUC].<br />
Tordylium maximum L. - Rusalca, sub platou<br />
prope littore Mare Nigrum, in fossa viam,<br />
7<br />
43º25′120″N, 28º31′125″E, alt. circa 12 m, 12 IV<br />
2008, G. Negrean (11.413) [BUC].<br />
Torilis nodosa (L.) Gaertner - Rusalca, ad littore<br />
Mare Nigrum, in herbosis prope marem,<br />
43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI<br />
2008, G. Negrean (10.784) [BUC].<br />
Erysiphe knautiae Duby, matrix:<br />
Knautia arvensis (L.) Coulter, Sofija S, prope<br />
Hotel Vitosha, in herbosis, 22 VI 2006, G. Negrean<br />
(7335) [BUC].<br />
Erysiphe lycopsidis R. Y. Zheng & G. Q.<br />
Chen, matrix:<br />
Anchusa arvensis (L.) Bieb. - Shabla E, ad littore<br />
Mare Nigrum, in locis herbosis, 43º24′954N″,<br />
28º30′061″E, alt. circa 10 m, 6 VI 2008, G.<br />
Negrean (10.641) [BUC].<br />
Erysiphe mougeotii (Lév.) <strong>de</strong> Bary, matrix:<br />
Lycium barbarum L. - Cavarna, centrum,<br />
43º26′13.44″N, 28º20′36.38″E, alt. circa 127 m, 23<br />
X 2008, G. Negrean (11.543) [BUC].<br />
Erysiphe polyphaga Hammarl. = Golovinomyces<br />
orontii (Castagne) V. P. Heliuta<br />
Erysiphe ranunculi Grev., matrix:<br />
Ranunculus constantinopolitanus (DC.) D’Urv. -<br />
Ecrene N, ad oram rivuli Batova, 43º21′10.43″N,<br />
28º04′31.74″E, alt. circa 2 m, 3 VI 2006, G.<br />
Negrean (7236) [BUC].<br />
Erysiphe thesii L. Junell, matrix:<br />
Thesium alpinum L., Sofija S, Montes Vitosha, in<br />
herbosis subalpinis, 23 VI 2006, G. Negrean (7347)<br />
[BUC].<br />
Erysiphe trifolii Grev., matrix:<br />
Medicago arabica L. - Camen Brjag, centrum, in<br />
locis ru<strong>de</strong>ralis, 43º27′20.83″N, 28º33′04.63″E, alt.<br />
circa 35 m, 23 X 2008, G. Negrean (11.531, A)<br />
[BUC].<br />
Melilotus officinalis (L.) Pallas - Cavarna SW,<br />
Bojorets S, Caliacria, in locis ru<strong>de</strong>ralis,<br />
43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22<br />
X 2008, G. Negrean (11.522) [BUC]. Sofija S,<br />
prope Hotel Moskva, in herbosis, 22 VI 2006, G.<br />
Negrean (7333) [BUC].<br />
Trifolium hybridum L. subsp. elegans (Savi)<br />
Aschers. & Graebn., Sofija S, Hortus Botanicus, in<br />
herbosis, 20 VI 2006, G. Negrean (7313) [BUC].<br />
Golovinomyces orontii (Castagne) V. P.<br />
Heliuta (Erysiphe polyphaga Hammarl.), matrix:
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Sedum sarmentosum Bunge - Camen Brjag, motel,<br />
cult., 43º27′20.40″N, 28º33′13.07″E, alt. circa 35<br />
m, 7 VI 2008, G. Negrean (10.697) [BUC].<br />
Neoerysiphe g<strong>ale</strong>opsidis (DC.) U. Braun<br />
(Erysiphe g<strong>ale</strong>opsidis DC.), matrix:<br />
Lamium amplexicaule L. Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300E, alt. circa 5 m, 9 V 2008, G. Negrean<br />
(10.434) [BUC]. Cavarna S, prope hotel, in locis<br />
ru<strong>de</strong>ralis, 43º24′50.69″N, 28º21′16.92″E, alt. circa<br />
20 m, 12 IV 2008, G. Negrean (10.250) [BUC].<br />
Balcic, centrum, ru<strong>de</strong>ral, 4 VI 2006, G. Negrean<br />
(7252) [BUC]. Sofija, centrum, 20 VI 2005, G.<br />
Negrean (6136) [BUC].<br />
Phyllactinia guttata (Wallr.: Fr.) Lév., matrix:<br />
Fraxinus angustifolia Vahl subsp. oxycarpa<br />
(Bieb. ex Willd.) Franco & Rocha Afonso -<br />
Cavarna, centrum, 43º26′13.44″N, 28º20′36.38″E,<br />
alt. circa 127 m, 23 X 2008, G. Negrean (11.549)<br />
[BUC].<br />
Podosphaera euphorbiae (Castagne) U. Braun<br />
& S. Takam., matrix:<br />
Euphorbia esula L. subsp. orientalis (Boiss.)<br />
Molero & Rovira, 1992 (Euphorbia esula subsp.<br />
tommasini-ana (Bertol.) Nyman) - Bălgarevo SE,<br />
Cap Caliacra, in herbosis, 43º23′02.13″N,<br />
28º26′53.26″E, alt. circa 70 m, 10 VIII 2008, G.<br />
Negrean (11.486) [BUC].<br />
Sawa<strong>de</strong>a bicornis (Wallr.: Fr.) Homma<br />
(Uncinula bicornis (Wallr.: Fr.) Lév., matrix:<br />
Acer negundo L., subspont., Sofija S, prope Univ.<br />
Technica, 21 VI 2006, G. Negrean (7319) [BUC].<br />
Sphaerotheca aphanis (Wallr.) U. Braun,<br />
matrix:<br />
Geum urbanum L., Sofija S, prope Hotel<br />
Moskva, 22 VI 2006, G. Negrean (7324) [BUC].<br />
Sphaerotheca fugax Penz. & Sacc., matrix:<br />
Erodium ciconium (L.) L’Hér. - Bălgarevo E, Cap<br />
Caliacra, in herebosis, 3 VI 2006, G. Negrean<br />
(7256) [BUC].<br />
Geranium rotundifolium L. - Bălgarevo E, Cap<br />
Caliacra, vers E, ut Mare Nigrum, in herbosis<br />
ru<strong>de</strong>ralis, 15 IV 2006, G. Negrean (7071a) [BUC].<br />
Bălgarevo SE, Vallis Bolata, ad saxa calcarea, solo<br />
terra-rossa, 43º22′59.77″N, 28º28′20.77″E, alt. circa<br />
15 m, 12 IV 2008, G. Negrean (10.267) [BUC].<br />
Taphrina <strong>de</strong>formans (Berk.) Tul., matrix:<br />
8<br />
Prunus dulcis Miller - Balcic, prope Hortus<br />
Botanicus, cult., 2 V 2008, G. Negrean (10.343)<br />
[BUC].<br />
Prunus persica (L.) Batsch - Bălgarevo E, Cap<br />
Caliacra, cult., 30 IV 2008, G. Negrean (10.358)<br />
[BUC].<br />
Taphrina pruni Tul., matrix:<br />
Prunus cerasifera Ehrh. - Shabla E, prope littore<br />
Mare Nigrum, 43º33′755″N, 28º35′250″E, alt. circa<br />
3 m, 9 V 2008, G. Negrean (11.112) [BUC].<br />
Prunus domestica L. - Balcic, prope hotel<br />
Eisberg, cult., 30 IV 2008, G. Negrean (10.359)<br />
[BUC].<br />
Venturia geranii (Fr.) G. Winter, matrix:<br />
Erodium ciconium (L.) L’Herit. - Duranculac E,<br />
ad littore Mare Nigrum, in locis ru<strong>de</strong>ralis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 11 IV<br />
2008, G. Negrean (10.224) [BUC], 9 V 2008, G.<br />
Negrean (10.430) [BUC].<br />
UREDINALES:<br />
Aecidium euphorbiae Link, O, I, matrix:<br />
Euphorbia agraria Bieb. - Bălgarevo E, ut Cap<br />
Caliacra, in herbosis, 14 IV 2006, G. Negrean<br />
(7064a) [BUC]. Bălgarevo E, Cap Caliacra, in<br />
herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa<br />
25 m, 12 IV 2008, G. Negrean (10.239 [BUC].<br />
Euphorbia myrsinites L. - Bălgarevo E, vallis<br />
Bolata Dere, terra rossa, 43º23′08.40″N,<br />
28º27′59.49″E, alt. circa 11 m, 12 IV 2008, G.<br />
Negrean (10.268) [BUC]. Bălgarevo E, Cap<br />
Caliacra, in herbosis, 43º22′982″N, 28º26′459″E,<br />
alt. circa 72 m, 12 IV 2008, G. Negrean (10.236)<br />
[BUC].<br />
Euphorbia nicaeensis All. s. l. - Crapetz, ut<br />
f<strong>ale</strong>za, in herbosis, 12 IV 2008, G. Negrean<br />
(10.240) [BUC].<br />
Euphorbia seguieriana Necker - Duranculac E, ad<br />
littore Mare Nigrum, in locis arenosis,<br />
43º41′908″N, 28 º34′300″E, alt. circa 5 m, 11 IV<br />
2008, G. Negrean (10.217) [BUC].<br />
Melampsora euphorbiae (Ficinus & C.<br />
Schub.) Castagne, matrix:<br />
Euphorbia helioscopia L. - Duranculac E, ad<br />
littore Mare Nigrum, in locis ru<strong>de</strong>ralis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V<br />
2008, G. Negrean (10.431, ii, iii) [BUC].<br />
Duranculac E, ad littore Mare Nigrum, in locis
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
ru<strong>de</strong>ralis, 43º41′908″N, 28º34′300″E, alt. circa 5 m,<br />
6 VI 2008, G. Negrean (10.598) [BUC]. Rusalca,<br />
sub platou prope littore Mare Nigrum, in herbosis,<br />
43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI<br />
2008, G. Negrean (10.661) [BUC]. Bălgarevo E,<br />
Cap Caliacra, in herbosis, 43º22′982″N,<br />
28º26′459″E, alt. circa 72 m, 12 IV 2008, G.<br />
Negrean (10.233, ii, iii) [BUC]. Bălgarevo E, inter<br />
Cap Caliacra et Bolata Dere, prope littore Mare<br />
Nigrum, in herbosis, 43º22′405-23′144″N,<br />
28º27′928-965″E, alt. circa 30 m, 11 V 2008, G.<br />
Negrean (10.497) [BUC]. Cavarna, sub Montes<br />
Cheracman, 30 IV 2008, G. Negrean (10.376)<br />
[BUC].<br />
Phragmidium mucronatum (Pers.) Schltdl,<br />
matrix:<br />
Rosa canina L. - Rusalca, prope littore Mare<br />
Nigrum, in herbosis et petrosis, 43º24′733″N,<br />
28º29′776″E, alt. circa 55 m, 10 V 2008, G.<br />
Negrean (10.462, i) [BUC].<br />
Phragmidium potentillae (Pers.) P. Karst.,<br />
matrix:<br />
Potentilla pedata Nestler - Bălgarevo E, Cap<br />
Caliacra, in herebosis, 4 VI 2006, G. Negrean<br />
(7294) [BUC].<br />
Potentilla taurica Willd. - Shabla E, ad littore<br />
Mare Nigrum, in locis herbosis, 43º24′954″N,<br />
28º30′061″E, alt. circa 10 m, 6 VI 2008, G.<br />
Negrean (10.638) [BUC].<br />
Phragmidium sanguisorbae (DC.) Schröt.,<br />
matrix:<br />
Sanguisorba minor Scop. s. l., Sofija S, prope<br />
Hotel Vitosha (N), ad viam ferream, 26 VI 2006, G.<br />
Negrean (7372) [BUC].<br />
Phragmidium violaceum (Schultz) G. Winter,<br />
matrix:<br />
Rubus candicans Weihe ex Rchb. - Bălgarevo E,<br />
Rusalca, in herbosis, supra Mare Nigrum,<br />
43º25′02.98″N, 28º30′50.05″E, alt. circa 20 m, 19<br />
VII 2008, G. Negrean (11.406) [BUC].<br />
Rubus discolor Weihe & Nees - Bălgarevo SE,<br />
prope littore Mare Nigrum, sub abruptum, prope<br />
ferma pisciculturae, in locis herbosis,<br />
43º25′02.83″N, 28º31′00.18″E, alt. circa 5 m, 10<br />
VIII 2008, G. Negrean (11.498, ii) [BUC].<br />
Puccinia allii (DC.) F. Rudolphi, matrix:<br />
Allium tauricum (Besser ex Rchb.) Grossh. –<br />
Bălgarevo E, Cap Caliacra, 43º22′982″N,<br />
9<br />
28º26′459″E, alt. circa 72 m, 12 IV 2008, G.<br />
Negrean (10.288) [BUC, ii].<br />
Allium sp. - Ecrene N, ad oram rivuli Batova, in<br />
herbosis, 43º20′55.87″N, 28º04′25.15″E, alt. circa 1<br />
m, 3 VI 2006, G. Negrean (7235) [BUC].<br />
Puccinia asperulae-cynanchicae Wurth,<br />
matrix:<br />
Asperula tenella Heuffel ex Boiss. - NE Bulgaria:<br />
prov. Burgas: Aitos, in petrosis, 11 VI 1973, G.<br />
Negrean [BUC].<br />
Puccinia calcitrapae DC., matrix:<br />
Carduus pycnocephalus L. - Rusalca, sub platou<br />
prope littore Mare Nigrum, in saxosis,<br />
43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI<br />
2008, G. Negrean (10.698, iii) [BUC].<br />
Centaurea thracica (Janka) Hayek - Bălgarevo E,<br />
<strong>de</strong>xtra vallis Bolata Dere, in herbosis, 43º23′144″N,<br />
28º27′965E″, alt. circa 34 m, 6 VI 2008, G.<br />
Negrean (10.785) [BUC].<br />
Puccinia cesatii J. Schröt., matrix:<br />
Dichanthium ischemum (L.) Roberty - Bălgarevo<br />
SE, Cap Caliacra, 43º23′02.13″N, 28º26′53.26″E,<br />
alt. circa 30 m, 10 VIII 2008, G. Negrean (11.483)<br />
[BUC].<br />
Puccinia crepidis J. Schröt., matrix:<br />
Crepis foetida L. subsp. rhoeadifolia (Bieb.)<br />
Čelak. - Bălgarevo SE, Cap Caliacra, in herbosis<br />
ru<strong>de</strong>ralis, prope Archer (Boris Caragea),<br />
43º21′38.78″N, 28º27′55.78″E, alt. circa 8 m, 11 IV<br />
2008, G. Negrean (10.231) [BUC]. Cap Caliacra, in<br />
herbosis, 43º22′03.97″N, 28º27′57.08″E , alt. circa<br />
30 m, 8 VI 2008, G. Negrean (10.753) [BUC].<br />
Puccinia dobrogensis Săvul. & O. Săvul. (?=<br />
Puccinia caucasica Savelli), matrix:<br />
Iris pumila L. - Bălgarevo E, Cap Caliacra, in<br />
herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa<br />
25 m, 8 VI 2008, G. Negrean (11.120) [BUC].<br />
Puccinia gladioli (Requien) Cast., I, matrix:<br />
V<strong>ale</strong>rianella costata (Steven) Betcke - Bălgarevo<br />
E, Cap Caliacra, situs archaeologicus, in herbosis,<br />
14 IV 2006, G. Negrean (7817) [BUC].<br />
V<strong>ale</strong>rianella sp. - Bălgarevo E, Cap Caliacra, in<br />
herbosis, 43º22′03.97″N, 28º27′57.08″E, alt. circa<br />
25 m, 12 IV 2008, G. Negrean (10.255, i) [BUC].<br />
Cavarna E, in herbosis, 13 IV 2008, G. Negrean<br />
(10.261) [BUC].<br />
Puccinia graminis DC., matrix:<br />
Festuca drymeja Mert. & Koch - distr. Shumen:<br />
Shumen W, Platous Shumen, in silvis,
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
43º14′55.96″N, 26º53′45.52″E, alt. circa 474 m, 18<br />
VII 2008, G. Negrean (11.603).<br />
Puccinia helianthi Schwein., matrix:<br />
Helianthus annuus L. „Florae Pleno”, cult. -<br />
Camen Briag, centrum, 43º27′20.83″N,<br />
28º33′04.63″E, alt. circa 35 m, 23 X 2008, G.<br />
Negrean (11.537) [BUC].<br />
Puccinia hieracii Mart., matrix:<br />
Hieracium bauhinii Besser, Sofija S, prope Hotel<br />
Vitosha, 22 VI 2006, G. Negrean, matrix conf.<br />
Krahuleć (Pruhonice) (7322) [BUC].<br />
Puccinia isiacae (Thüm.) G. Wint., matrix:<br />
Cardaria draba (L.) Desv. subsp. draba -<br />
Duranculac E, ad littore Mare Nigrum, in ru<strong>de</strong>ratis,<br />
43º41′908″N, 28 º34′300″E, alt. circa 5 m, 9 V<br />
2008, G. Negrean (10.426, i) [BUC].<br />
Puccinia komarovii Tranzschel, matrix:<br />
Impatiens parviflora DC. - Sofija S, Hotel<br />
Vitosha (N), Parc, subspont. ad viam ferream, 24<br />
VI 2006, G. Negrean (7380) [BUC; SOM]. Fungus<br />
adventivus novus Bulgariae. Originally from<br />
Central Asia, alien in Europe. In Romania on<br />
Impatiens parviflora DC., subspont. in Botanical<br />
Gar<strong>de</strong>n of Cluj-Napoca, V<strong>ale</strong>a Pârîul Ţiganilor,<br />
46°51′46″N, 23°35′20″E, alt. 347 m, 5 VII 1993, G.<br />
Negrean [BUCM 129.306].<br />
Puccinia lapsanae Fuckel, matrix:<br />
Lapsana communis L. - Sofija S, prope Hotel<br />
Vitosha (N), ad viam ferream, 24 VI 2006, G.<br />
Negrean (7374) [BUC].<br />
Puccinia malvacearum Bertero ex Mont. – iii,<br />
matrix:<br />
Althaea hirsuta L. - Duranculac E, ad littore<br />
Mare Nigrum, in locis herbosis, 43º40′289″N,<br />
28º33′922″E, alt. circa 5 m, 6 VI 2008, G. Negrean<br />
(11.121) [BUC]. Bălgarevo E, Cap Caliacra, in<br />
herbosis, 43º22′405″N, 28º27′928″E, alt. circa 30<br />
m, 8 VI 2008, G. Negrean (10.767) [BUC].<br />
Malva sylvestris L. - Shabla E, ad littore Mare<br />
Nigrum, in locis herbosis, 43º24′954″N,<br />
28º30′061″E, alt. circa 10 m, 9 V 2008, G. Negrean<br />
(10.444) [BUC]. Yailata, prope littore Mare<br />
Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt.<br />
circa 25 m, 10 V 2008, G. Negrean (11.123)<br />
[BUC]. Rusalca NNE, in herbosis, 43º25′34.94″N,<br />
28º31′51.46″E, alt. circa 8 m, 12 IV 2008, G.<br />
Negrean (10.276) [BUC], 43º24′733″N,<br />
28º29′776″E, alt. circa 65 m, 10 V 2008, G.<br />
Negrean (10.460) [BUC], Rusalca, ad littore Mare<br />
10<br />
Nigrum, in locis herbosis, 43º24′954″N,<br />
28º30′061″E, alt. circa 10 m, 6 VI 2008, G.<br />
Negrean () [BUC]. Balcic, centrum, in cortis<br />
moscheii, ru<strong>de</strong>ral, 13 IV 2006, G. Negrean (7044)<br />
[BUC]. Nesebăr, in arenosis ru<strong>de</strong>ralis ad littore<br />
Mare Nigrum, 5 VI 2006, G. Negrean (7439)<br />
[BUC].<br />
Puccinia minussensis Thüm., matrix:<br />
Lactuca tatarica (L.) C. A. Meyer - Duranculac<br />
E, ad littore Mare Nigrum, in ru<strong>de</strong>ratis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 9 V<br />
2008, G. Negrean (10.429) [BUC], 6 VI 2008, G.<br />
Negrean (10.598) [BUC].<br />
Puccinia pachyphloea Syd. & H. Syd., matrix:<br />
Rumex tuberosus L. subsp. tuberosus - Bălgarevo<br />
E, Cap Caliacra N, Bolata-Dere, in herbosis, 4 VI<br />
2006, G. Negrean (7396) [BUC].<br />
Puccinia pelargonii-zonalis Doidge, matrix:<br />
Pelargonium ×hortorum auct. - Sofija S, Hortus<br />
Botanicus, in caldaria, cult. 42º43′..N, 23º19′...E, 20<br />
VI 2006, G. Negrean (7311) [BUC; SOM].<br />
Puccinia phragmitis (Schumach.) Körn.,<br />
matrix:<br />
Rumex patientia L. s. l. - Duranculac E, ad littore<br />
Mare Nigrum, in ru<strong>de</strong>ratis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, G.<br />
Negrean (10.285, i) [BUC].<br />
Puccinia pimpinellae (F. Strauss) Link,<br />
matrix:<br />
Pimpinella peregrina L. - Yailata, prope littore<br />
Mare Nigrum, in abruptis, 43º26′552″N,<br />
28º32′930″E, alt. circa 25 m, 10 V 2008, G.<br />
Negrean (10.465) [BUC]. Rusalca, sub platou prope<br />
littore Mare Nigrum, in herbosis, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 7 VI 2008, G.<br />
Negrean (10.665) [BUC].<br />
Puccinia procera Dietel & Holw., matrix:<br />
Leymus racemosus (Lam.) Tzvelev subsp.<br />
sabulosus (Beb.) Tzvelev - Shabla E, ad littore<br />
Mare Nigrum, in locis herbosis, 43º33′755″N,<br />
28º35′250″E, alt. circa 3 m, 6 VI 2008, G. Negrean<br />
(10.639, ii) [BUC].<br />
Puccinia punctata Link, matrix:<br />
Galium verum L. subsp. verum - Bălgarevo SE,<br />
prope littore Mare Nigrum, sub abruptum, prope<br />
ferma pisciculturae, in locis herbosis,<br />
43º25′02.83″N, 28º31′00.18″E, alt. circa 5 m, 10<br />
VIII 2008, G. Negrean (11.505) [BUC].<br />
Puccinia recondita Dietel & Holw., matrix:
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Aegilops cylindrica Host - Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean<br />
(10.601) [BUC]. Balcic W, Cap Caliacra, in<br />
herbosis, 3 VI 2006, G. Negrean (7298) [BUC].<br />
Sofija S, prope Hotel Vitosha (N), ru<strong>de</strong>ral, 24 VI<br />
2006, G. Negrean (7377).<br />
Aegilops geniculata Roth - Rusalca, platou prope<br />
littore Mare Nigrum, in locis herbosis et petrosis,<br />
43º24′733″N, 28º29′776″E, alt. circa 60 m, 7 VI<br />
2008, G. Negrean (10.696, iii) [BUC]. Bălgarevo E,<br />
Cap Caliacra, in herbosis, 43º22′405″N,<br />
28º27′928″E, alt. circa 40 m, 8 VI 2008, G.<br />
Negrean (10.765, iii) [BUC].<br />
Anchusa sp. - Bălgarevo E, Cap Caliacra, in<br />
herbosis, 43º22′982″N, 28º26′459″E, alt. circa 72<br />
m, 12 IV 2008, G. Negrean (10.234) [BUC].<br />
Bromus sterilis L. subsp. elegans - Sofija S,<br />
prope Hotel Vitosha, 22 VI 2006, G. Negrean<br />
(7317) [BUC].<br />
Echium italicum L. subsp. pyramidatum (DC.) . -<br />
Bălgarevo SE, Cap Caliacra, 43º23′02.13″N,<br />
28º26′53.26″E, alt. circa 70 m, 10 VIII 2008, G.<br />
Negrean (11.482, i) [BUC].<br />
Clematis vitalba L.- Yailata, prope littore Mare<br />
Nigrum, in abruptis, 43º26′552″N, 28º32′930″E, alt.<br />
circa 25 m, 10 V 2008, G. Negrean (10.777, i)<br />
[BUC]. Bălgarevo E, inter Cap Caliacra et Bolata<br />
Dere, prope littore Mare Nigrum, in herbosis,<br />
43º22′405-23′144″N, 28º27′928-965″E, alt. circa 50<br />
m, 11 V 2008, G. Negrean (10.506) [BUC]. Ecrene<br />
N, ad oram rivuli Batova, in arenosis,<br />
43º21′10.43″N, 28º04′31.74″E, alt. circa 2 m, 3 VI<br />
2006, G. Negrean (7238) [BUC].<br />
Puccinia sii-falcariae J. Schröt., matrix:<br />
Falcaria vulgaris Bernh. - Duranculac E, ad<br />
littore Mare Nigrum, in locis ru<strong>de</strong>ralis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI<br />
2008, G. Negrean (10.597) [BUC]. Shabla E, ad<br />
littore Mare Nigrum, in locis herbosis,<br />
43º24′954″N, 28º30′061″E, alt. circa 10 m, 9 V<br />
2008, G. Negrean (10.441) [BUC]. Cavarna E, in<br />
herbosis, 12 IV 2008, G. Negrean (10.256) [BUC].<br />
Bălgarevo E, inter Cap Caliacra et Bolata Dere,<br />
prope littore Mare Nigrum, in herbosis, 43º22′405-<br />
23′144″N, 28º27′928-965″E, alt. circa 30 m, 11 V<br />
2008, G. Negrean (10.497) [BUC]. Bălgarevo E,<br />
Cap Caliacra N, Bolata-Dere, in herbosis, 6 VI<br />
2006, G. Negrean (7396) [BUC].<br />
11<br />
Puccinia tanaceti DC., matrix:<br />
Artemisia absinthium L. - Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 6 VI 2008, G. Negrean<br />
(10.594) [BUC]. Cavarna SW, Caliacria, in locis<br />
ru<strong>de</strong>ralis, 43º25′03.47″N, 28º16′48.25″E, alt. circa<br />
46 m, 23 X 2008, G. Negrean (11.520) [BUC].<br />
Tranzschelia pruni-spinosae (Pers.) Dietel –<br />
(ii), iii, matrix:<br />
Prunus cerasifera Ehrh. - Duranculac NE, ad<br />
confines Bulgariae, 43º44′10.05″N, 28º33′24.68″E,<br />
alt. circa 27 m, 23 X 2008, G. Negrean (11.559)<br />
[BUC; SOM].<br />
Uromyces dianthi (Pers.: Pers.) Niessl, matrix:<br />
Dianthus leptopetalus Willd. - Bălgarevo E,<br />
sinistra vallis Bolata Dere, prope littore Mare<br />
Nigrum, in herbosis, 43º23′140″N, 28º28′000″E,<br />
alt. circa 35 m, 20 VII 2008, G. Negrean (11.476)<br />
[BUC].<br />
Petrorhagia prolifera (L.) P. W. Ball &<br />
Heywood - Bălgarevo E, Cap Caliacra, in herbosis,<br />
4 VI 2006, G. Negrean (7256) [BUC].<br />
Uromyces limonii (DC.) Lév., matrix:<br />
Limonium latifolium (Sm.) Kuntze - Yailata, ad<br />
littore Mare Nigrum, 43º26′131″N, 28º32′665″E,<br />
alt. circa 12 m, 19 VII 2008, G. Negrean (11.444)<br />
[BUC].<br />
Limonium meyeri (Boiss.) Kuntze - Rusalca, sub<br />
platou, prope littore Mare Nigrum, in saxosis,<br />
43º25′116″N, 28º31′126″E, alt. circa 10 m, 7 VI<br />
2008, G. Negrean (10.690) [BUC]. Rusalca, sub<br />
platou, prope littore Mare Nigrum, in saxosis,<br />
43º25′02.83″N, 28º31′00.18″E, alt. circa 10 m, 19<br />
VII 2008, G. Negrean (11.434) [BUC].<br />
Uromyces lineolatus (Desm.) Schroet., matrix:<br />
Scirpus maritimus L. subsp. maritimus - Shabla<br />
NE, ad littore Mare Nigrum, in arenosis,<br />
43º24′954″N, 28º30′061″E, alt. circa 4 m, 20 VII<br />
2008, G. Negrean (11.578) [BUC].<br />
Uromyce punctatus J. Schröt., matrix:<br />
Astragalus cornutus Pallas - Bălgarevo E, <strong>de</strong>xtra<br />
vallis Bolata Dere, prope littore Mare Nigrum, in<br />
herbosis, 43º23′140″N, 28º28′000″E, alt. circa 35<br />
m, 20 VII 2008, G. Negrean (11.449) [BUC].<br />
Uromyces rumicis (Schumach.) G. Winter,<br />
matrix:<br />
Rumex patientia L. s. l. - Camen Briag, centrum,<br />
in locis ru<strong>de</strong>ralis, 43º27′20.83″N, 28º33′04.63″E,
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
alt. circa 35 m, 23 X 2008, G. Negrean (11.530)<br />
[BUC].<br />
Uromyces scutellatus (Pers.: Pers.) Lév.,<br />
matrix:<br />
Euphorbia agraria Bieb. - Crapetz, ut f<strong>ale</strong>za, in<br />
herbosis, 12 IV 2008, G. Negrean (10.288) [BUC].<br />
Euphorbia dobrogensis Prodan - Duranculac E,<br />
ad littore Mare Nigrum, in herbosis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 9 V 2008, G. Negrean<br />
(11.113) [BUC].<br />
Euphorbia nicaeensis All. s.l. - Duranculac E, ad<br />
littore Mare Nigrum, in herbosis et petrosis,<br />
43º41′908″N, 28º34′300″E, alt. circa 5 m, 6 VI<br />
2008, G. Negrean (10.599) [BUC].<br />
Euphorbia seguieriana Necker - Sofija S, prope<br />
Hotel Vitosha, 22 VI 2006, G. Negrean (7320).<br />
Uromyces trifolii-repentis Liro, matrix:<br />
Trifolium hybridum L. subsp. elegans (Savi)<br />
Aschers. & Graebn., Sofija S, Hortus Botanicus, in<br />
herbosis, 20 VI 2006, G. Negrean (7310) [BUC].<br />
USTOMYCETES<br />
Entyloma c<strong>ale</strong>ndulae (Ou<strong>de</strong>m.) <strong>de</strong> Bary,<br />
matrix:<br />
C<strong>ale</strong>ndula officinalis L., Sofija S, cult., 22 VI<br />
2006, G. Negrean (7320).<br />
Microbotryum violaceoverrucosum<br />
(Bran<strong>de</strong>nb. & Schwinn) Vánky, matrix:<br />
Silene bupleuroi<strong>de</strong>s Sm. s. l. - Yailata, platou<br />
prope littore Mare Nigrum, in saxosis,<br />
43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII<br />
2008, G. Negrean (11.470) [BUC].<br />
Microbotryum violaceum (Pers.: Pers.) G.<br />
Deml & Oberw. s. l., matrix:<br />
Silene latifolia Poiret subsp. alba (Miller) Greuter<br />
& Bur<strong>de</strong>t - Shabla E, ad littore Mare Nigrum, in<br />
locis herbosis, 43º24′954″N, 28º30′061″E, alt. circa<br />
10 m, 9 V 2008, G. Negrean (11.116) [BUC; CL].<br />
Camen Brjag, centrum, in cortis, 43º27′20.83″N,<br />
28º33′04.63″E, alt. circa 35 m, 23 X 2008, G.<br />
Negrean (11.538) [BUC; CL].<br />
Sorosporium saponariae F. Rudolphi, matrix:<br />
Silene bupleuroi<strong>de</strong>s Sm. s. l. - Yailata, platou<br />
prope littore Mare Nigrum, in saxosis,<br />
43º26′552″N, 28º32′930″E, alt. circa 25 m, 8 VIII<br />
2008, G. Negrean (11.471) [BUC].<br />
Ustilago cynodontis (Pass.) P. Henn., matrix:<br />
12<br />
Cynodon dactylon (L.) Pers. - Shabla NE, ad littore<br />
Mare Nigrum, in arenosis, 43º24′954N,<br />
28º30′061″E, alt. circa 4 m, 20 VII 2008, G.<br />
Negrean (11.584) [BUC]. Rusalca, sub platou prope<br />
littore Mare Nigrum, in Paliuretum, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 19 VII 2008, G.<br />
Negrean (11.417) [BUC]. Bălgarevo E, <strong>de</strong>xtra<br />
vallis Bolata Dere, in herbosis, 43º23′144″N,<br />
28º27′965″E, alt. circa 34 m, 6 VI 2008, G.<br />
Negrean (10.782) [BUC].<br />
Ustilago ornithogali (J. C. Schmidt & Kunze)<br />
J. G. Kühn, matrix:<br />
Gagea pusilla (F. W. Schmidt) Schult. & Schult.<br />
fil. - Cavarna E, in herbosis, 43º24′25.14″N,<br />
28º22′19.22″E, alt. circa 60 m, 12 IV 2008, G.<br />
Negrean (11.444) [BUC].<br />
Ustilago vaillantii L.-R. Tul. & C. Tul., matrix:<br />
Scilla bithynica Boiss. - Ecrene N, ad oram rivuli<br />
Batova, in Alnetum, Fraxinetum pallisiae et<br />
Salicetum, in locis humidis, 43º20′55.58″N,<br />
28º04′06.51″E, alt. circa 2 m, 13 IV 2006, G.<br />
Negrean (7056) [BUC; CL].<br />
AGARICALES, POLYPORALES, GASTEROMYCETALES<br />
Dendrothele acerina (Pers.: Fr.) P. A. Lemke,<br />
matrix:<br />
Acer campestre L. - Rusalca, sub platou, prope<br />
littore Mare Nigrum, in silvis, 43º25′140″N,<br />
28º31′120″E, alt. circa 15 m, 7 VI 2008, G.<br />
Negrean (10.700) [BUC].<br />
Fomitopsis pinicola (Sw.) P. Karst., matrix:<br />
Picea abies (L.) Karsten subsp. abies - Montes<br />
Vitosha, 23 VI 2006, G. Negrean (7824) [BUC].<br />
Hymenochaete rubiginosa (Dicks.) Lév.,<br />
matrix:<br />
Quercus robur L. - Sofija, Hotel Vitosha N, Hotel<br />
Moskva W, park, 24 VI 2006, G. Negrean (7389c)<br />
[BUC].<br />
Lepista panaeolus (Fr.) P. Karsten - ad solum,<br />
Dobrogea, Cavarna SW, Bojorets S, Caliac-<br />
ria, in locis ru<strong>de</strong>ralis, 43º25′03.47″N, 28º16′48.25″E,<br />
alt. circa 46 m, 23 X 2008, G. Negrean (12.077).<br />
Polyporus melanopus (Pers.) Fr., matrix: in<br />
lignos, distr. Shumen: Shumen W, Platous Shumen,<br />
in Fagetum, 43º14′55.96″N, 26º53′45.52″E,<br />
alt. circa 474 m, 18 VII 2008, G. Negrean (11.604)<br />
[BUC].
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Polyporus varius Pers.: Fr., matrix: ad ramulos<br />
<strong>de</strong>cidous, distr. Shumen: Shumen, park,<br />
43º16′12.73″N, 26º560′30.79″E, alt. circa 205 m,<br />
18 VI 2008, G. Negrean (11.586) [BUC].<br />
Suillus bellinii (Inzenga) Watling, ad solum,<br />
sub Pinus nigra Arnold, cult., Dobrogea, Cavarna<br />
SW, Bojorets S, Caliacria, in abruptis et petrosis,<br />
43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22<br />
X 2008, G. Negrean (11.592) [BUC; CL].<br />
Trametes hirsuta (Wulfen) Pilát, matrix: in<br />
lignos, distr. Shumen: Shumen W, Platous<br />
Shumen, in Fagetum, 43º14′55.96″N,<br />
26º53′45.52″E, alt. circa 474 m, 18 VII 2008, G.<br />
Negrean (11.605) [BUC].<br />
Tulostoma brum<strong>ale</strong> Pers.: Pers., ad solum,<br />
Duranculac E, ad littore Mare Nigrum, in arenosis<br />
maritimis, 43º40′296″N, 28º33′918″E, alt. circa 5<br />
m, 9 V 2008, comm. P. Anastasiu, <strong>de</strong>t. G. Negrean<br />
& P. Anastasiu (10.426) [BUC].<br />
Tulostoma squamosum Pers. - Ecrene N, ad<br />
oram rivuli Batova, in arenosis, 43º21′10.43″N,<br />
28º04′31.74″E, alt. circa 2 m, 13 IV 2006, G.<br />
Negrean (7054) [BUC].<br />
Volvariella gloiocephala (DC.: Fr.) Boekhout<br />
& En<strong>de</strong>rle - ad solum, Dobrogea, Cavarna SW,<br />
Bojorets S, Caliacria, in locis ru<strong>de</strong>ralis,<br />
43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 23<br />
X 2008, G. Negrean (12.075).<br />
FUNGI ANAMORPHICI<br />
Ampelomyces quisqualis Ces.,<br />
socio cum: Erysiphe cruciferarum Opiz ex<br />
L. Junell - matrix:<br />
Alyssum hirsutum Bieb. - Duranculac E, ad littore<br />
Mare Nigrum, in locis ru<strong>de</strong>ralis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, G.<br />
Negrean (10.283) [BUC].<br />
Socio cum: Erysiphe cynoglossi (Wallr.) U. Braun,<br />
matrix:<br />
Echium italicum L. subsp. pyramidatum (DC.) . -<br />
Bălgarevo SE, Cap Caliacra, in herbosis,<br />
43º23′02.13″N, 28º26′53.26″E, alt. circa 70 m, 10<br />
VIII 2008, G. Negrean (11.481) [BUC].<br />
Cercospora plumbaginea Sacc. & D. Sacc.,<br />
matrix:<br />
Plumbago europaea L. - Cavarna S, prope hotel,<br />
in locis ru<strong>de</strong>ralis, 43º21′17.41″N, 28º21′17.41″E,<br />
13<br />
alt. circa 30 m, 10 VIII 2008, G. Negrean (11.487)<br />
[BUC].<br />
Cercospora taurica Tranzsch., matrix:<br />
Heliotropium europaeum L. - Camen Briag,<br />
centrum, in locis ru<strong>de</strong>ralis, 43º27′20.83″N,<br />
28º33′04.63″E, alt. circa 35 m, 23 X 2008, G.<br />
Negrean (11.591) [BUC; CL ]. Cavarna SW,<br />
Bojorets S, Caliacria, in locis ru<strong>de</strong>ralis,<br />
43º25′03.47″N, 28º16′48.25″E, alt. circa 46 m, 22<br />
X 2008, G. Negrean (11.529) [BUC].<br />
Napicladium celtidis Cavara, matrix:<br />
Celtis australis L. - Cavarna S, prope hotel, in<br />
locis ru<strong>de</strong>ralis, 43º21′17.41″N, 28º21′17.41″E, alt.<br />
circa 30 m, 10 VIII 2008, G. Negrean (11.489)<br />
[BUC].<br />
Ovularia obliqua (Cooke) Ou<strong>de</strong>m., matrix:<br />
Rumex patientia L. s. l. - Duranculac E, ad littore<br />
Mare Nigrum, locis ru<strong>de</strong>ratis, 43º41′908″N,<br />
28º34′300″E, alt. circa 5 m, 11 IV 2008, G.<br />
Negrean (10.218) [BUC].<br />
Ramularia arvensis Sacc., matrix:<br />
Potentilla recta L., Sofija S, prope Hotel Vitosha,<br />
20 VI 2006, G. Negrean (7309) [BUC].<br />
Ramularia beticola Fautrey & Lambotte,<br />
matrix:<br />
Beta trigyna Waldst. & Kit. - Rusalca, sub platou<br />
prope littore Mare Nigrum, 43º24′750″N,<br />
28º29′790″E, alt. circa 15 m, 10 V 2008, G.<br />
Negrean (10.464) [BUC].<br />
Ramularia centaureae Lindr., matrix:<br />
Centaurea salonitana Vis. - Bălgarevo E, Cap<br />
Caliacra, in herbosis, 43º22′982″N, 28º26′459″E,<br />
alt. circa 72 m, 8 VI 2008, G. Negrean (10.754)<br />
[BUC].<br />
Ramularia libanotidis Bubák, matrix:<br />
Seseli campestre Besser - Rusalca, sub platou,<br />
prope littore Mare Nigrum, 43º25′116″N,<br />
28º31′126″E, alt. circa 10 m, 7 VI 2008, G.<br />
Negrean (10.693) [BUC].<br />
Ramularia ranunculi-oxyspermi Lobik,<br />
matrix:<br />
Ranunculus oxyspermus Bieb. - Cavarna E, in<br />
herbosis, 43º24′25.14″N, 28º22′19.22″E, alt. circa<br />
60 m, 12 IV 2008, G. Negrean (10.262) [BUC].<br />
5. References<br />
[1] NEGREAN Gavril, CONSTANTINESCU<br />
Ovidiu & DENCHEV Cvetomir M. 2004.
Limitative mycotic factors from some plants.../ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
Addition to the Peronospr<strong>ale</strong>s of Bulgaria.<br />
Mycologia Balcanica 1(1): 69-72.<br />
[2] NEGREAN Gavril & DENCHEV Cvetomir M.<br />
2000. New record of Bulgarian parasitic fungi.<br />
Flora Mediterranea (P<strong>ale</strong>rmo) 10: 101-108.<br />
[3] NEGREAN Gavril & DENCHEV Cvetomir M.<br />
2002. New record of fungi from bulgarian<br />
Dobrudzha. Pp. 21-24. In: N. RANDJELOVIĆ<br />
(ed.), Proceedings of Sixth Symposium on Flora<br />
of Southeastern Serbia and Adjacent Territories,<br />
July 4-7, 2000, Sokobanja, Yugoslavia. Vuk<br />
Karadžić, Niš.<br />
[4] NEGREAN Gavril & DENCHEV Cvetomir M.<br />
2004. Addition to the Erysiph<strong>ale</strong>s of Bulgaria.<br />
Mycologia Balcanica 1(1): 63-66.<br />
[5] BRAUN Uwe. 1987. A monograph of the<br />
Erysiph<strong>ale</strong>s (pow<strong>de</strong>ry mil<strong>de</strong>ws). Beih. Nova<br />
Hedw. Heft 89. Berlin, Stuttgart: J. Cramer, 700<br />
pp., 316 fig.<br />
[6] BRAUN Uwe. 1995. The pow<strong>de</strong>ry mil<strong>de</strong>ws<br />
(Erysiph<strong>ale</strong>s) of Europe. Jena: Gustav Fischer<br />
Verlag, i-iv, 1-337 pp., ill. 112, ISBN 3-334-<br />
60994-4 (HB).<br />
[7] DENCHEV Cvetomir M. 1995. Bulgarian<br />
Uredin<strong>ale</strong>s. Mycotaxon 55: 405-465.<br />
[8] FAKIROVA Violeta Ilieva ● ФАКИРОВА<br />
Виолета Илиева. 1991. Fungi Bulgaricae 1<br />
tomus ordo Erysiph<strong>ale</strong>s ● Гъбите в България 1<br />
том разред Еrysiph<strong>ale</strong>s, red. princip. prof. Dr. I.<br />
Kovachevsky; edit. Simeon Vanev, Edit. Acad.<br />
Bulgaricae, Sofija, 154 pp.<br />
[9] MAJEWSKI T. 1977. Grzyby (Mycota), T. IX,<br />
Podstawczaki (Basidiomycetes), Rdzawniko we<br />
(Uredin<strong>ale</strong>s) I, Flora Polska, Warsawa - Kraków:<br />
Panstwowe Wydawnictwo Naukowe. 396 pp.<br />
[10] MAJEWSKI T. 1979. Grzyby (Mycota), T. XI,<br />
Podstawczaki (Basidiomycetes), Rdzawnikowe<br />
(Uredin<strong>ale</strong>s) II, Flora Polska, Warsawa -<br />
Kraków: Panstwowe Wydawnictwo Naukowe.<br />
463 pp. + Erata + 2 Pl.<br />
[11] SĂVULESCU T. 1953. Monografia<br />
Uredin<strong>ale</strong>lor din Republica Populară Română ●<br />
Monographia Uredinalium Reipublicae<br />
Popularis Romanicae, vol. 1-2. Bucureşti: Edit.<br />
Aca<strong>de</strong>miei Române, 1166 pp. (vol. 1: 1-332 + ixxiv<br />
+ liii Pl. + 21 Tab.; vol. 2: 333-1168. /B:<br />
339-343/.<br />
[12] SĂVULESCU T. 1957. Ustilagin<strong>ale</strong>le din<br />
Republica Populară Romînă ● Ustilagin<strong>ale</strong>s<br />
14<br />
Reipublicae Popularis Romanicae, vol. 1-2.<br />
Bucureşti: Edit. Aca<strong>de</strong>miei Romîne, 1168 pp.<br />
/vol. I: 1-545 pp; vol. II: 546-1170 pp., in<strong>de</strong>x:<br />
1141-1168/.<br />
[13] SCHOLLER M. 1996. Die Erysiph<strong>ale</strong>s,<br />
Puccini<strong>ale</strong>s und Ustilagin<strong>ale</strong>s <strong>de</strong>r Vorpommerschen<br />
Bod<strong>de</strong>nlandschaft - Ökologisch-floristiche,<br />
florengeschichtliche und morphologisch-taxonomische<br />
Untersuchungen. Regensb. Mykol. Schriften<br />
6: 1-325.<br />
[14] VANEV Simeon Georgiev, DIMITROVA<br />
Evtimia Georgieva & ILIEVA Elena Ivanova ●<br />
ВАНЕВ Симеон Георгиев, ДИМИТРОВА<br />
Евтимия Георгиева & ИЛИЕВА Елена<br />
Иванова. 1993. Fungi Bulgaricae 2 tomus ordo<br />
Peronospor<strong>ale</strong>s ● Гъбите в България 2 Tом<br />
разред Peronospor<strong>ale</strong>s. Red. principali Prof. Dr.<br />
Ivan KOVACHEVSKI, Editit tomum, Violeta<br />
FAKI-ROVA. Sofija: Edit. Aca<strong>de</strong>miae<br />
Scientiarum Bulgaricae, 195 pp. + Erata, 1 fig., 1<br />
tab., 57 pl. ISBN 954-430-227-1 (t. 2).<br />
[15] SĂVULESCU T. (ed.). 1952-1976. Flora<br />
României ● Flora Romaniae. Bucureşti: Edit.<br />
Aca<strong>de</strong>miei Române. Vol. 1-13.<br />
[16] TUTIN T. G., BURGES N. A., CHATER A.<br />
O., EDMONDSON J. R., HEYWOOD V. H.,<br />
MOORE D. M., VALENTINE D. H., WALTERS<br />
S. M. & WEBB D. A. (eds, assist. by<br />
J. R. AKEROYD & M. E. NEWTON;<br />
appendices ed. by R. R. MILL). 1993. Flora<br />
Europaea. 2nd ed. Vol. 1. Psilotaceae to<br />
Platanaceae. Cambridge: Cambridge University<br />
Press xlvi, 581 pp., illus. ISBN 0-521-41007-X<br />
(HB).<br />
[17] TUTIN T. G., HEYWOOD V. H., BURGES<br />
N. A., MOORE D. M., VALENTINE D. H.,<br />
WALTERS S. M. & WEBB D. A. (eds). 1964-<br />
1980. Flora Europaea. Vols. 1-5. Cambridge:<br />
Cambridge University Press.<br />
[18] HOLMGREN Patricia K. & HOLMGREN<br />
Noel H. (ed.). 1992. Plant specialists in<strong>de</strong>x: In<strong>de</strong>x<br />
to specialists in the systematics of plants and<br />
fungi based on data from In<strong>de</strong>x Herbariorum<br />
(Herbaria), edition 8. Königstein: Koeltz<br />
Scientific Books, 1-394. [Regnum Vegetabile<br />
120], ISBN 3-87429-331-9 (HB).<br />
[19] NEGREAN G. 1992. Violeta Ilieva Fakirova,<br />
Fungi Bulgaricae 1 tomus ordo Erysiph<strong>ale</strong>s, red.
Gavril Negrean/ Ovidius University Annals of Biology-Ecology 14: 3-15 (2010)<br />
princip. prof. Dr. I. Kovachevsky; edit. Simeon<br />
Vanev, Edit. Acad. Bulgaricae, Sofija, 1991, 154<br />
pp.etc. Stud. Cerc. Biol., Ser. Biol. Veg. 44(2):<br />
196-197. /recenzie critică/.<br />
15<br />
Aknowledgments<br />
We thanks Mrs. Professor Paulina Anastasiu<br />
for the help given in or<strong>de</strong>r to draw this material and<br />
to Dr. Krahulec (Pruhonice) for confirming the<br />
i<strong>de</strong>ntification of Hieracium bauhinii.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE MEDICINAL PLANTS OF PROVADIISKO PLATEAU<br />
Dimcho ZAHARIEV, Desislav DIMITROV<br />
University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences,<br />
115 Universitetska Str., 9712, Shumen, Bulgaria,<br />
dimtchoz@yahoo.com<br />
_________________________________________________________________________________________<br />
Abstract: Consi<strong>de</strong>rable taxonomical diversity of the medicinal plants of Provadiisko Plateau is established: 376<br />
species of vascular plants from 261 genera and 86 families. Most families (77.91%) and genera (98.85%) are<br />
represented in small numbers – 1 to 4. The analysis of their life form indicates that the geophytes dominante,<br />
followed by the groups of the phanerophytes and the hemi cryptophytes. These biological types are represented<br />
mainly by perennial herbaceous plants (53.19%) and annual herbaceous plants (12.77%). The largest percentage<br />
species are of the circumboreal type (36.17%). Among the medicinal plants, there are 4 en<strong>de</strong>mites and 29 relicts.<br />
39 species with protection statute are <strong>de</strong>scribed. The anthropophytes among the medicinal plants are 236 species<br />
(62.77%).<br />
Keywords: Provadiisko Plateau, medicinal plants, analysis of medicinal plants, protected species.<br />
______________________________________________________________________________________<br />
1. Introduction<br />
In physiographic terms the Provadiisko Plateau<br />
belongs to the Danube hilly plain area, i.e. the<br />
Ludogorsko-Provadiiska subarea [1]. The Northern<br />
plateau bor<strong>de</strong>r is the Provadiiska River; in the East it<br />
reaches to the Devnya Valley; in the South, the<br />
Provadiisko Plateau is separated from Roiaksko<br />
Plateau by Glavnica River; and finally, west of the<br />
Provaddisko Plateau is the Shumensko Plateau. The<br />
average altitu<strong>de</strong> is 250 m. above sea level. The<br />
highest point is Sakartepe in the western parts of the<br />
plateau with its height of 389 m. The plateau is<br />
located in the Transcontinental climate region, district<br />
Dobrudjansko Plateau [2]. Winds are coming mostly<br />
from the North and Northeast. The average annual<br />
temperature is around 12°С. The average monthly<br />
temperatures are always positive. The temperature in<br />
January is the lowest (1.2°С) and in July – the highest<br />
(22.6°С). The minimum temperature rarely fall to<br />
18°С, and the average maximum temperature reaches<br />
27°С. The maximum rainfalls are in May and June<br />
and the minimum – in March and September. The<br />
annual amount of rainfalls is around 530 mm.<br />
Average humidity is around 76-77%; lowest in the<br />
summer (70%) and highest in the winter (82%) [3].<br />
The soils, according to the FAO classification, are<br />
two types. The first type is calcic chernozems located<br />
on the slopes and in the areas with low slope. The<br />
second type is calvaric fluvisols located in the<br />
Provadiiska Valley [4].<br />
In terms of its flora, the plateau belongs to the<br />
region of Northeastern Bulgaria. The vegetation<br />
inclu<strong>de</strong>s: forests of Carpinus betulus L. and Quercus<br />
cerris L., partly with Carpinus orientalis Mill.; mixed<br />
forests of Carpinus betulus L. and Quercus cerris L.,<br />
partly with Quercus d<strong>ale</strong>champii Ten., Acer<br />
campestre L., etc.; mixed forests of Tilia tomentosa<br />
Moench., with Carpinus betulus L. or Quercus cerris<br />
L., partly also with Quercus d<strong>ale</strong>champii Ten., Acer<br />
campestre L., etc.; forest and shrubs of Carpineta<br />
orientalis; mixed forests of Quercus cerris L.,<br />
Quercus pubescens Willd. and Cotinus coggygria<br />
Scop., partly with a secondary prev<strong>ale</strong>nce of Cotinus<br />
coggygria Scop.; mixed forests of Fraxinus ornus L.<br />
and Carpinus orientalis Mill., partly of secondary<br />
origin; shrubs with prev<strong>ale</strong>nce of Paliureta spinachristi,<br />
combined with xerothermal frass communities<br />
mostly replacing xerothermal forest communities of<br />
Quercus cerris L. and Quercus frainetto Ten.; shrub<br />
and grass steppe and xerothermal communities;<br />
xerothermal grass communities with a prev<strong>ale</strong>nce of<br />
Dichantieta ischaemi, Poaeta bulbosae, Poaeta<br />
concinnae, Chrysopogoneta grylli and Ephemereta;<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
mesoxerothermal grass vegetation with a prev<strong>ale</strong>nce<br />
of Poa bulbosa L., Loium perenne L., Cynodon<br />
dactylon (L.) Pers., partly also Dichantium<br />
ischaemum (L.) Roberty and rarely Chrysopogon<br />
gryllus (L.) Trin., mostly in the village com<br />
monlands; mesophytous grass communities<br />
(meadows), replacing forests of Ulmus minor Mill.,<br />
Fraxinus oxicarpa Willd., Quercus robur L.,<br />
Quercus pedunculiflora C. Koch.; farm areas,<br />
replacing forests of Fagus sylvatica ssp. moesiaca<br />
(K. Maly) Hyelmq.; farm areas, replacing forests of<br />
Quercus d<strong>ale</strong>champii Ten.; farm areas, replacing<br />
forests of Ulmus minor Mill., Fraxinus oxicarpa<br />
Willd., Quercus pedunculiflora C. Koch. [5].<br />
The first studies of the flora of the plateau have<br />
been conducted in the 1990s by Vasil Kovachev<br />
around Madara, Kaspitchan and Provadia [6]. The<br />
results are found in the first volume on Bulgarian<br />
flora [7] and its supplement [8]. Hermengild Shkorpil<br />
also conducted botanical researchin the vicinity of<br />
Provadia in the early twentieth century [6].<br />
So far, data on the medicinal plants in the area of<br />
Provadiisko Plateau have been published by authors<br />
for the the territory of Municipality Provadia [9] and<br />
by Zahariev and Uzunov for the protected area<br />
Madarski rock wreaths [10].<br />
The Provadiisko Plateau is a part of the protected<br />
zone Provadiisko-Roiaksko Plateau by Natura 2000,<br />
according to Council Directive 92/43/EEC of the<br />
European Community to protect natural habitats and<br />
of wild fauna and flora [11].<br />
2. Material and Methods<br />
The field studies were conducted on the route<br />
method in 2007-2009. The names of the taxons are<br />
taken from the Flora of PR Bulgaria, Vol. І – Х [12].<br />
The update of the taxons is consistent with APG II<br />
[13]. The life forms are presented by Raunkier [14].<br />
In their <strong>de</strong>termination was used Flora of PR Bulgaria,<br />
Vol. І – Х [12]. The biological types are presented by<br />
Kozuharov [15]. The floristic elements and en<strong>de</strong>mites<br />
are presented by Asiov et all. [16]. The relicts are<br />
presented by Gruev and Kuzmanov [17], Peev [18],<br />
Boža et all. [19], Peev et all. [20]. The protection<br />
status is presented using the following documents:<br />
Council Directive 92/43/EEC of the European<br />
18<br />
Community to protect natural habitats and of wild<br />
fauna and flora [11], Convention on<br />
International Tra<strong>de</strong> in Endangered Species of Wild<br />
Fauna and Flora (CITES) [21], Red book of PR<br />
Bulgaria [22], IUCN Red List for Bulgaria [23],<br />
Biological Diversity Act [24], Or<strong>de</strong>r for special<br />
arrangements for the conservation and use of<br />
medicinal plants [25]. The anthropophytes are<br />
presented by Stefanov and Kitanov [26].<br />
3. Results and Discussions<br />
As a result of the research of the medicinal plants<br />
of the Provadiisko Plateau 376 species of vascular<br />
plants from 261 genera and 86 families have been<br />
in<strong>de</strong>tified. They represent 9.83% from all species,<br />
29.36% from all genera and 50.89% from all plant<br />
families in Bulgaria.<br />
Most families (77.91%) and genera (98.85%) are<br />
represented in small numbers: 1 to 4.<br />
Almost all families (86.05%) are represented<br />
with 1-4 genera. Only 13.95% from the families<br />
inclu<strong>de</strong>d 5 or more genera (Table 1). Most genera are<br />
found in the families: Asteraceae (28), Lamiaceae<br />
(22), Fabaceae (21), Rosaceae (15), Apiaceae (14)<br />
and Brassicaceae (12).<br />
Table 1. Families with greatest number of genera<br />
Families Genera<br />
Asteraceae 28<br />
Lamiaceae 22<br />
Fabaceae 21<br />
Rosaceae 15<br />
Apiaceae 14<br />
Brassicaceae 12<br />
Scrophulariaceae 8<br />
Ranunculaceae 8<br />
Caryophyllaceae 8<br />
Boraginaceae 7<br />
Poaceae 5<br />
Solanaceae 5<br />
Most families – 77.91% have 1-4 species. Only<br />
22.09% of the families are represented by 5 or more<br />
species (Table 2). Most species belong to the<br />
following families: Asteraceae (42), Lamiaceae (41),
Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
Fabaceae (28), Rosaceae (26), Brassicaceae (15),<br />
Apiaceae (15), Ranunculaceae (12) and<br />
Scrophulariaceae (11).<br />
Almost all genera (98.85%) are represented by 1-<br />
4 species. Most species – more than 5 have only<br />
1.15% of the genera (Table 3): Centaurea, Geranium<br />
and Thymus.<br />
Table 2. Families with greatest number of species<br />
Families Species<br />
Asteraceae 42<br />
Lamiaceae 41<br />
Fabaceae 28<br />
Rosaceae 26<br />
Brassicaceae 15<br />
Apiaceae 15<br />
Ranunculaceae 12<br />
Scrophulariaceae 11<br />
Boraginaceae 9<br />
Caryophyllaceae 9<br />
Orchidaceae 7<br />
Geraniaceae 6<br />
Polygonaceae 6<br />
Solanaceae 6<br />
Aspleniaceae 5<br />
Oleaceae 5<br />
Poaceae 5<br />
Rubiaceae 5<br />
Salicaceae 5<br />
Table 3. Genera with greatest number of species<br />
Families Genera Species<br />
Asteraceae Centaurea 5<br />
Geraniaceae Geranium 5<br />
Lamiaceae Thymus 5<br />
In the analysis of the life forms were obtained the<br />
following results (Table 4): The phanerophytes (Ph)<br />
are represented by 86 species. The megaphanerophytes<br />
are represented by 10 species, the most<br />
common of which are: Acer pseudoplatanus L.,<br />
Fraxinus excelsior L., Gleditsia triacanthos L., Pinus<br />
sylvestris L., Quercus frainetto Ten., Quercus robur<br />
L.<br />
19<br />
The mezophanerophytes are 35 species, of which<br />
essential are: Acer campestre L., Acer<br />
pseudoplatanus L., Carpinus betulus L., Fagus<br />
sylvatica L., Fraxinus ornus L., Tilia tomentosa<br />
Moench, Ulmus minor Mill.<br />
The microphanerophytes are 27 species, the most<br />
common of which are: Acer tataricum L., Cornus<br />
mas L., Corylus avellana L., Cotinus coggygria<br />
Scop., Crataegus monogyna Jacq., He<strong>de</strong>ra helix L.,<br />
Ligustrum vulgare L., Paliurus spina-christi Mill.,<br />
Prunus spinosa L., Rosa canina L., Rubus caesius L.,<br />
Sambucus nigra L.<br />
The nanophanerophytes are 11 species, which<br />
are essential: Clematis vitalba L., Genista tinctoria<br />
L., Teucrium chamaedrys L., Teucrium polium L.<br />
The succulents are represented by 3 species:<br />
Sedum acre L., Sedum album L. and Sedum<br />
maximum (L.) Suter.<br />
Table 4. Life forms<br />
Group Subgroup Species<br />
Megaphanerophytes 10<br />
Mezophanerophytes 35<br />
Phanerophytes Microphanerophytes 27<br />
(Ph) Nanophanerophytes 11<br />
Epiphytes –<br />
Succulents 3<br />
Hamephytes (Ch) 5<br />
Hemi cryptophytes (H) 65<br />
Therophyte – hemi cryptophytes 45<br />
(Th-H)<br />
Cryptophytes Geophytes 126<br />
(Cr)<br />
Helophytes 1<br />
Hydrophytes –<br />
Therophytes (Th) 48<br />
The group of hamephytes (Ch) inclu<strong>de</strong>s 5<br />
species: Dictamnus albus L., Ruscus aculeatus L.,<br />
Satureia montana L., Thymus jankae Čelak., Thymus<br />
zygioi<strong>de</strong>s Griseb.<br />
The hemi cryptophytes (H) are 65 species, of<br />
which most common are: Agrimonia eupatoria L.,<br />
Carlina vulgaris L., Cichorium intybus L.,<br />
Clinopodium vulgare L., Echium vulgare L.,<br />
Eryngium campestre L., Lotus corniculatus L.,<br />
Marrubium peregrinum L., Plantago lanceolata L.,
The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
Plantago media L., Polygala major Jacq.,<br />
Ranunculus ficaria L., Salvia nemorosa L., Silene<br />
vulgaris (Moench) Garcke, Taraxacum officin<strong>ale</strong><br />
Web., Trifolium pratense L., Trifolium repens L.,<br />
Verbena officinalis L., Viola odorata L.<br />
The transition group therophytes – hemi<br />
cryptophytes (Th-H) comprises 45 species, of which<br />
essential are: Alliaria petiolata (Bieb.) Cavara et<br />
Gran<strong>de</strong>, Arctium lappa L., Capsella bursa-pastoris<br />
Moench., Daucus carota L., Erodium cicutarium (L.)<br />
L̀ Her., Heracleum sibiricum L., Malva sylvestris L.,<br />
Plantago major L., Stellaria media (L.) Vill.,<br />
Tordylium maximum L., Verbascum <strong>de</strong>nsiflorum<br />
Bertol., Viola tricolor L.<br />
The group of cryptophytes (Cr) is the largest and<br />
inclu<strong>de</strong>s 127 species. Their significant proportion can<br />
be explained by the dominance of forest habitats<br />
within the plateau. Geophytes dominate with total of<br />
126 species; the most wi<strong>de</strong>spread of them are:<br />
Achillea millefolium L., Anemone ranunculoi<strong>de</strong>s L.,<br />
Artemisia absinthium L., Artemisia vulgaris L.,<br />
Chelidonium majus L., Convolvulus arvensis L.,<br />
Coronilla varia L., Fragaria vesca L., Galanthus<br />
elwesii Hook. fil., Galanthus nivalis L., Geum<br />
urbanum L., Isopyrum thalictroi<strong>de</strong>s L., Potentilla<br />
argentea L., Sanguisorba minor Scop., Scilla bifolia<br />
L., Urtica dioica L. The helophytes is represented by<br />
one species only: Typha latifolia L.<br />
The therophytes (Th) are 48 species. The most<br />
wi<strong>de</strong>spread are: Galium aparine L., Lactuca serriola<br />
L., Lamium purpureum L., Lolium temulentum L.,<br />
Melilotus officinalis (L.) Pall., Papaver rhoeas L.,<br />
Parietaria lusitanica L., Xeranthemum annuum L.<br />
The largest group species in terms of biological<br />
types (Figure 1) are perennial plants (p) – 200 species<br />
(53.19%). Their dominance can be explained with the<br />
wi<strong>de</strong> variety of communities and habitats within the<br />
plateau.<br />
The annual plants (a) are 48 species (12.77%),<br />
which can be explained by the presence of dry rocky<br />
terrain and arable lands on the plateau.<br />
The tree species (t) are 39 (10.37%). The next<br />
group inclu<strong>de</strong>s shrubs (sh) – 29 species (7.71%). The<br />
transition group from annual to biennial plants (a-b)<br />
inclu<strong>de</strong>s 19 species (5.05%). The biennial plants (b)<br />
are 13 species (3.46%). There are species from<br />
transition group from tree to shrubs (sh-t) with 12<br />
20<br />
species (3.19%) and species from transition group<br />
from biennial to perennial plants (b-p) – 11 species<br />
(2.92%). The largest group inclu<strong>de</strong>s annual as well as<br />
perennial plants (a-p) and is represented by 5 species<br />
(1.33%).<br />
29<br />
12<br />
39<br />
200<br />
Fig. 1. Biological types<br />
The specific physiographic conditions on the<br />
Provadiisko Plateau <strong>de</strong>termined consi<strong>de</strong>rable<br />
diversity of floristic elements. 7 different types of<br />
floristic elements are established (Table 5). The<br />
dominant elements are elements from circumboreal<br />
type – 136 species (36.17%), followed by European<br />
elements – 101 species (26.86%) and Mediterranean<br />
elements – 67 species (17.82%). The en<strong>de</strong>mic<br />
component is represented by 4 species (1.06%). It<br />
inclu<strong>de</strong>s 3 Balkan en<strong>de</strong>mites – Achillea clypeolata<br />
Sibth. et Sm., Aesculus hippocastanum L., Inula<br />
aschersoniana Janka and 1 Balkan suben<strong>de</strong>mite –<br />
Syringa vulgaris L.<br />
Table 5. Floristic elements<br />
Floristic elements Species<br />
Circumboreal type 136<br />
European type 101<br />
Mediterranean type 67<br />
Pontic type 27<br />
Adventive type 20<br />
Cosmopolitan type 19<br />
Balkan en<strong>de</strong>mic and<br />
suben<strong>de</strong>mic type<br />
4<br />
Other 2<br />
This distribution can be explained by the location<br />
of the plateau in the transcontinental climate region.<br />
The proximity of the plateau to the bor<strong>de</strong>r of a<br />
48<br />
19 5 13<br />
11<br />
a<br />
a-b<br />
a-p<br />
b<br />
b-p<br />
p<br />
sh<br />
sh-t<br />
t
Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
temperate region is the reason for the prev<strong>ale</strong>nce of<br />
circumboreal and European floristic elements. At the<br />
same time, the impact of the continentalmediterranean<br />
region in terms of the Black Sea and<br />
the karst topography create conditions for the<br />
<strong>de</strong>velopment of a large number of mediterranean<br />
species.<br />
The flora of the plateau inclu<strong>de</strong>s significant<br />
number of relict species: 29. They account for 7.71%<br />
of the total number of species. The majority of the<br />
relict species are Tertiary relicts. They are 28 species:<br />
Abies alba Mil., Acer campestre L., Acer<br />
pseudoplatanus L., Acer tataricum L., Aesculus<br />
hippocastanum L., Betula pendula Roth, Carpinus<br />
betulus L., Carpinus orientalis Mill., Celtis australis<br />
L., Cercis siliquastrum L., Clematis vitalba L.,<br />
Corylus avellana L., Cotinus coggygria Scop.,<br />
Fraxinus excelsior L., Fraxinus ornus L., He<strong>de</strong>ra<br />
helix L., Juniperus communis L., Picea abies (L.)<br />
Karsten, Pinus nigra Arn., Populus alba L., Populus<br />
nigra L., Ruscus aculeatus L., Salix alba L., Salix<br />
caprea L., Smilax excelsa L., Taxus baccata L.,<br />
Ulmus minor Mill., Viscum album L. One of the relict<br />
species is quaternary – Galanthus nivalis L.<br />
39 species with protection statute are <strong>de</strong>scribed.<br />
One of them – Himantoglossum caprinum (Bieb.) C.<br />
Koch., is inclu<strong>de</strong>d in the list of species, protected by<br />
the Berne Convention and Natura 2000. In CITES 10<br />
species are inclu<strong>de</strong>d: Adonis vernalis L., Anacamptis<br />
pyramidalis C. Rich., Galanthus elwesii Hook. fil.,<br />
Galanthus nivalis L., Himantoglossum caprinum<br />
(Bieb.) C. Koch., Orchis morio L., Orchis purpurea<br />
Huds., Orchis simia L., Orchis tri<strong>de</strong>ntata Scop.,<br />
Platanthera chlorantha (Cust.) Rchb. In the IUCN<br />
Red List for Bulgaria 5 species are inclu<strong>de</strong>d un<strong>de</strong>r the<br />
category „Threatened”: Aesculus hippocastanum L.,<br />
Galanthus elwesii Hook. fil., Galanthus nivalis L.,<br />
Juniperus sabina L., Taxus baccata L., 2 species are<br />
inclu<strong>de</strong>d un<strong>de</strong>r the category „Vulnerable”:<br />
Anacamptis pyramidalis C. Rich., Himantoglossum<br />
caprinum (Bieb.) C. Koch, 2 species are in the<br />
category „Nearly threatened”: Anemone sylvestris L.,<br />
Cercis siliquastrum L. and 1 species is inclu<strong>de</strong>d in<br />
the category „Of least concern”: Tilia rubra DC. In<br />
the Red book for Bulgaria 4 species are inclu<strong>de</strong>d in<br />
the category „Endangered”: Aesculus hippocastanum<br />
L., Anemone sylvestris L., Galanthus nivalis L.,<br />
Taxus baccata L. and 4 species are inclu<strong>de</strong>d in the<br />
21<br />
category „Rare”: Artemisia pontica L., Cercis<br />
siliquastrum L., Juniperus sabina L., Tilia rubra DC.<br />
In the Biological Diversity Act 8 species are inclu<strong>de</strong>d<br />
in the category „Protected”: Aesculus hippocastanum<br />
L., Anacamptis pyramidalis C. Rich., Anemone<br />
sylvestris L., Galanthus elwesii Hook. fil., Galanthus<br />
nivalis L., Himantoglossum caprinum (Bieb.) C.<br />
Koch., Juniperus sabina L., Taxus baccata L. In the<br />
category “Un<strong>de</strong>r the protection and regulated use of<br />
nature” are 14 species: Asparagus officinalis L.,<br />
Crocus pallasii Bieb., Echinops sphaerocephalos L.,<br />
Gypsophila paniculata L., Helichrysum areanrium<br />
(L.) Moench., Lilium martagon L., Orchis morio L.,<br />
Orchis purpurea Huds., Orchis simia L., Orchis<br />
tri<strong>de</strong>ntata Scop., Polygonatum odoratum (Mill.)<br />
Druce, Ruscus aculeatus L., Salix caprea L., Scilla<br />
bifolia L. Collecting herbs is prohibited from the<br />
natural habitats of 15 species: Adonis vernalis L.,<br />
Althaea officinalis L., Artemisia santonicum L.,<br />
Asarum europaeum L., Asplenium trichomanes L.,<br />
Convallaria majalis L., Glaucium flavum Crantz,<br />
Helichrysum areanrium (L.) Moench., Orchis morio<br />
L., Orchis purpurea Huds., Orchis simia L., Orchis<br />
tri<strong>de</strong>ntata Scop., Phyllitis scolopendrium (L.)<br />
Newm., Ruscus aculeatus L., V<strong>ale</strong>riana officinalis L.<br />
Un<strong>de</strong>r a restrictive regime are 4 species: Berberis<br />
vulgaris L., Carlina acanthifolia All., Galium<br />
odoratum (L.) Scop., Sedum acre L.<br />
The anthropophytes among the medicinal plants<br />
are 236 species (62.77%). Many of them are<br />
consi<strong>de</strong>red weed or ru<strong>de</strong>ral plants. The most common<br />
as weed are: Anagallis arvensis L., Brassica nigra<br />
(L.) Koch, Centaurea cyanus L., Chenopodium<br />
album L., Chenopodium polyspermum L., Consolida<br />
hispanica (Costa) Greut. et Bur<strong>de</strong>t, Consolida regalis<br />
S. F. Gray, Cynodon dactylon (L.) Pers., Datura<br />
stramonium L., Myosotis arvensis (L.) Hill, Nigella<br />
arvensis L., Papaver rhoeas L., Senecio vulgaris L.,<br />
Stellaria media (L.) Vill., Thlaspi arvense L.,<br />
Xanthium strumarium L. Оf the ru<strong>de</strong>ral plants most<br />
common are: Capsella bursa-pastoris Moench.,<br />
Cardaria draba (L.) Desv., Chamomilla recutita (L.)<br />
Rausch., Chelidonium majus L., Conium maculatum<br />
L., Conyza cana<strong>de</strong>nsis (L.) Cronq., Heracleum<br />
sibiricum L., Lactuca serriola L., Parietaria<br />
lusitanica L., Sambucus ebulus L., Solanum<br />
dulcamara L., Urtica dioica L.
The medicinal plants of the Provadiisko Plateau / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
4. Conclusions<br />
[3] KOPRALEV, I. (main ed.), 2002. Geography of<br />
Bulgaria. Physical and socio-economic<br />
Consi<strong>de</strong>rable taxonomical diversity of the geography, Institute of Geography, BAS,<br />
medicinal plants of Provadiisko Plateau is i<strong>de</strong>ntified: Farkom, Sofia, 760 pp.<br />
376 species of vascular plants from 261 genera and [4] NINOV, N., 2002. Soils, in Kopr<strong>ale</strong>v, I. (main<br />
86 families.<br />
ed.). Geography of Bulgaria. Physical and socioeconomic<br />
geography, Institute of Geography,<br />
BAS, Farkom, Sofia, 760 pp.<br />
Most families (77.91%) and genera (98.85%) are<br />
represented in small numbers: 1 to 4.<br />
The analysis of life forms indicates the<br />
predominnce of geophytes, followed by the groups<br />
phanerophytes and hemi cryptophytes.<br />
The biological types are represented mainly by<br />
perennial herbaceous plants (53.19%) and annual<br />
herbaceous plants (12.77%).<br />
The i<strong>de</strong>ntified medicinal plants can be<br />
categorized into 7 types of floristic elements. The<br />
highest percentage species are of the circumboreal<br />
type (36.17%).<br />
Among the medicinal plants of Provadiisko<br />
Plateau 4 en<strong>de</strong>mites and 29 relicts are <strong>de</strong>scribed.<br />
39 species with protection status are <strong>de</strong>scribed:<br />
the use of 1 species is restricted by the Berne<br />
Convention and Natura 2000; 10 species are inclu<strong>de</strong>d<br />
in CITES; 10 species are inclu<strong>de</strong>d in IUCN Red List<br />
for Bulgaria; 8 species appear in the Red book for<br />
Bulgaria; 22 species are inclu<strong>de</strong>d in the Biological<br />
Diversity Act; 14 species are inclu<strong>de</strong>d in the category<br />
“Un<strong>de</strong>r the protection and regulated use of nature”,<br />
the collecting of herbs from their natural habitats is<br />
prohibited for 15 species, and 4 species are un<strong>de</strong>r a<br />
restrictive regime.<br />
The anthropophytes among the medicinal plants<br />
are 236 species (62.77%). Many of them are<br />
consi<strong>de</strong>red weed or ru<strong>de</strong>ral plants.<br />
5. References<br />
[1] GALABOV, J., 1966. Main lines of the relief<br />
(Common morphographic and morphometric<br />
characteristics), in Geography of Bulgaria,<br />
Physical geography – Relief, Vol. 1, Sofia.<br />
[2] VELEV, S., 2002. Climatic zoning, in Kopr<strong>ale</strong>v,<br />
I. (main ed.). Geography of Bulgaria. Physical<br />
and socio-economic geography, Institute of<br />
Geography, BAS, Farkom, Sofia, 760 pp.<br />
22<br />
[5] BONDEV, I., 1991. The vegetation of Bulgaria.<br />
Map in М 1:600 000 with explanatory text,<br />
University Press St. Kliment Ohridski, Sofia, 183<br />
pp.<br />
[6] STANEV, S., 2001. Little known names from<br />
Bulgarian botany, Pensoft, Sofia – Moscow, 202<br />
pp.<br />
[7] VELENOVSKY, J., 1891. Flora Bulgarica, Praga,<br />
676 рp.<br />
[8] VELENOVSKY, J., 1898. Flora Bulgarica,<br />
Supplementum I, Praga, 420 рp.<br />
[9] ZAHARIEV, D., Dimitrov D., 2009. The<br />
medicinal plants in area of Provadiisko Plato<br />
(Municipality Provadia), 8th National conference<br />
with international participation „Natural sciences<br />
– 2009”, 2-3.10.2009, Varna (upcoming).<br />
[10] ZAHARIEV, D., Uzunov G., 2009. A study of<br />
the flora in Protected place Madarski skalni<br />
venci, 8th National conference with international<br />
participation „Natural sciences – 2009”, 2-<br />
3.10.2009, Varna (upcoming).<br />
[11] Council Directive 92/43/EEC of the European<br />
Community to protect natural habitats and of<br />
wild fauna and flora.<br />
[12] Flora of PR Bulgaria, Vol. І-Х, 1963-1995,<br />
Publishing House of BAS, Sofia.<br />
[13] CHASE, M. (corresponding author), 2003. An<br />
update of the Angiosperm Phylogeny Group<br />
classification for the or<strong>de</strong>rs and families of<br />
flowering plants: APG II, The Linnean Society of<br />
London, Botanical Journal of the Linnean<br />
Society, 141: 399–436.<br />
[14] PAVLOV, D., 2006. Phytocoenology,<br />
Publishing House of University of Forestry,<br />
Sofia, 251 pp.<br />
[15] KOZUHAROV, S. (ed.), 1992. I<strong>de</strong>ntifier of the<br />
vascular plants in Bulgatia, Nauka i izkustvo,<br />
Sofia, 788 pp.
Dimcho Zahariev, Desislav Dimitrov / Ovidius University Annals, Biology-Ecology Series 14: 17-23 (2010)<br />
[16] ASIOV B., Petrova A., Dimitrov D., Vasilev R.,<br />
2006. Conspectus of the Bulgarian vascular flora.<br />
Distribution maps and floristic elements,<br />
Bulgarian Biodiversity Foundation, Sofia, 452<br />
pp.<br />
[17] GRUEV, B., Kuzmanov B., 1994 – General<br />
biogeography, University Press St. Kliment<br />
Ohridski, Sofia, 498 pp.<br />
[18] PEEV, D., 2001. National park Rila.<br />
Management plan 2001 – 2010. Adopted by<br />
Resolution №522 of Council of Ministers on<br />
04.07.2001, Sofia, 338 pp.<br />
[19] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić<br />
D., 2005. Flora “Rimskog šanca” (Vojvodina,<br />
Srbija), 8th Symposium on the flora of<br />
Southeastern Serbia and Neighbouring Regions,<br />
Niš, 20-24.06.2005, Abstracts, рр. 55.<br />
[20] PEEV, D., Kozuharov S., Anchev M., Petrova<br />
A., Ivanova D., Tzoneva S., 1998. Biodiversity<br />
of Vascular Plants in Bulgaria, In: Curt Meine<br />
(ed.), Bulgaria's Biological Diversity:<br />
Conservation Status and Needs Assessment,<br />
Volumes I and II, Washington, D.C.,<br />
Biodiversity Support Program, pp. 55–88.<br />
[21] Convention on International Tra<strong>de</strong> in<br />
Endangered Species of Wild Fauna and Flora,<br />
State Gazette number 6 from 21 Januari 1992.<br />
[22] Red book of PR Bulgaria, Vol. 1, Plants, 1984,<br />
Publishing House of BAS, Sofia, 447 pp.<br />
[23] PETROVA А., Vladimirov V. (eds.), 2009. Red<br />
List of Bulgarian vascular plants, Phytologia<br />
Balcanica 15 (1): 63–94.<br />
[24] Biological Diversity Act, State Gazette number<br />
77 from 9 august 2002, pp. 9–42. Amen<strong>de</strong>d in<br />
State Gazette number 94 from 16 November<br />
2007.<br />
[25] Or<strong>de</strong>r number RD-72 from 3 februari 2006 for<br />
special arrangements for the conservation and<br />
use of medicinal plants, State Gazette number 16<br />
from 21 Februari 2006.<br />
[26] STEFANOV, B., Kitanov B., 1962. Kultigenen<br />
plants and kultigenen vegetation in Bulgaria,<br />
Publishing House of BAS, Sofia, 275 pp.<br />
23
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE PLANTS WITH PROTECTION STATUTE, ENDEMITES AND RELICTS<br />
OF THE SHUMENSKO PLATEAU<br />
Dimcho ZAHARIEV, Elka RADOSLAVOVA<br />
University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences,<br />
115 Universitetska Str., 9712, Shumen, Bulgaria<br />
dimtchoz@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: As a result of our investigations of the Shoumen Plateau in the period 1998-2009, 786 species were<br />
i<strong>de</strong>ntified, of which the number of species with conservation status is 80 (10.18%). 2 of those species are<br />
inclu<strong>de</strong>d in Appendix II of Directive 92/43/ЕЕС. 24 of the species are inclu<strong>de</strong>d in CITES. 32 species are<br />
inclu<strong>de</strong>d in the IUCN Red List for Bulgaria un<strong>de</strong>r the following categories: threatened – 13, vulnerable – 9,<br />
nearly threatened – 5 and least concern – 5 species. In the Red book for Bulgaria, there are 7 endangered species<br />
and 14 are rare plants. In the Biological Diversity Act, 23 species are inclu<strong>de</strong>d in Appendix 3 and further 28<br />
species – in Appendix 4. The collecting of herbs from their natural habitats is prohibited for 12 species, and 6<br />
species are un<strong>de</strong>r a restriction. 29 species (3.69%) are en<strong>de</strong>mites. These are 17 Balkan suben<strong>de</strong>mites, 9 Balkan<br />
en<strong>de</strong>mites and 3 Bulgarian en<strong>de</strong>mites. The flora of the plateau inclu<strong>de</strong>s a significant number of relict species –<br />
42. (5.34%). The majority of them, 39 species, are Tertiary relicts, 2 are quaternary relicts and 1 is a postglacial<br />
steppe relict.<br />
Keywords: Shumensko Plateau, plants with protection statute, en<strong>de</strong>mites, relicts.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Shumensko Plateau refers to an area in the hills<br />
east of the Danube plain, which was <strong>de</strong>clared<br />
protected by Natura 2000. This was <strong>de</strong>termined by the<br />
hills’ role in support of the biodiversity among large<br />
territories of scattered forests. The majority of the<br />
Shumensko Plateau area – 3929.9 ha (53%), was<br />
<strong>de</strong>clared for National Park in 1980. In 2003, the park<br />
was recognized as Nature Park. The regime of use<br />
and management of the park is <strong>de</strong>termined by the<br />
Protected Areas Act [1] and the Management Plan for<br />
the Nature Park [2].<br />
In the park is located the Bukaka Preserve. This<br />
is a forest area of 63.04 ha, <strong>de</strong>clared protected due to<br />
the indigenous forest that has existed there for several<br />
centuries and is comprised of Fagus sylvatica subsp.<br />
moesiaca. On the territory of the preserve, all human<br />
activity is prohibited, except for people passing on<br />
specifically marked paths.<br />
Shumensko Plateau has been <strong>de</strong>clared protected<br />
by Natura 2000 and its estimated area is 4490.62 hа.<br />
This territory is also protected un<strong>de</strong>r the Council<br />
Directive 92/43/EEC of the European Community<br />
for protecting natural habitats of wild fauna and flora<br />
[3].<br />
The unique combination of conditions in terms<br />
of topography, water resources, climate and soil,<br />
<strong>de</strong>termine the diversity of the plant species in the<br />
area. In the past, Velenovsky and his collaborators<br />
Hermengild Shkorpil and Anani Iavashev began the<br />
study of the plateau’s flora. In the 1980s, they<br />
collected the first botanical data in Northeast<br />
Bulgaria, including the area of the Shumen vicinity<br />
[4]. Their research is presented in the first volume on<br />
the Bulgarian flora [5] and its supplement [6].<br />
Davidov [7] conducted his own research on the flora<br />
of Shumen and the territory around the town. Further<br />
information about individual species, distributed on<br />
the plateau, can be found in Stoyanov and Stefanov<br />
[8, 9, 10], Stoyanov, Stefanov and Kitanov [11] and<br />
in Flora of PR Bulgaria, Vol. І – Х [12]. The<br />
diversity of species of the Orchidaceae family has<br />
been studied by Radoslavova [13]. In the<br />
Management Plan for the National Park Shumensko<br />
Plateau [2]: there are 550 species of vascular plants<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The plants with protection statute, en<strong>de</strong>mites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
(i.e. mosses not counted) <strong>de</strong>scribed in that source. Our<br />
studies [14] show that the number of vascular plants<br />
on the territory of the entire plateau is 786 species.<br />
According to the forest <strong>de</strong>velopment project of<br />
the Shumen Forestry [15], a total of 16 species have<br />
conservation statute and are inclu<strong>de</strong>d in the Red book<br />
of PR Bulgaria [16]. Seven of the species are<br />
endangered: Aesculus hippocastanum L., Anacamptis<br />
pyramidalis C. Rich., Anemone sylvestris L.,<br />
Castanea sativa Mill., Galanthus nivalis L.,<br />
Himantoglossum hircinum (L.) Spreng., Paeonia<br />
tenuifolia L. Nine of the species are rare: Atropa<br />
belladonna L., Celtis caucasica Willd., Cercis<br />
siliquastrum L., Cyclamen coum Mill., Fibigia<br />
clypeata (L.) Medic., Fritillaria pontica Wahl.,<br />
Haplophyllum thesioi<strong>de</strong>s G. Don., Jurinea le<strong>de</strong>borii<br />
Bunge., Pastinaca umbrosa Stev. ex DC. Six of these<br />
species are protected by the Biological Diversity Act<br />
(BDA) [17]: Aesculus hippocastanum L., Anacamptis<br />
pyramidalis C. Rich., Anemone sylvestris L.,<br />
Cyclamen coum Mill., Galanthus nivalis L.,<br />
Himantoglossum hircinum (L.) Spreng.<br />
Six of the plateau species are listed in the Red<br />
Book of the district Shumen [18]. Two of them are<br />
endangered: Lilium martagon L. and Campanula<br />
euxina (Vel.) Ancev. Four of the species are rare:<br />
Himantoglossum hircinum (L.) Spreng., Anacamptis<br />
piramidalis (L.) Rich., Ruscus hyppoglosum L. and<br />
Galium pasch<strong>ale</strong> Forsskal.<br />
In the Management Plan of the National Park<br />
Shumensko Plateau [2] are found 18 species with<br />
conservation statute that are also listed in the Red<br />
book of PR Bulgaria. Five of them are in the category<br />
“endangered”: Anemone sylvestris L., Colchicum<br />
davidovii Stefanov, Galanthus nivalis L., Ruta<br />
graveolens L., Veronica spicata L. Thirteen species<br />
fall into the category “rare”: Anthemis regis-borisii<br />
Stoj. et Acht., Anthemis rumelica (Velen.) Stoj. et<br />
Acht., Celtis caucasica Willd., Cyclamen coum Mill.,<br />
Erodium hoefftianum C. A. Meyer, Fibigia clypeata<br />
(L.) Medic., Fritillaria graeca Boiss. & Spruner,<br />
Fritillaria pontica Wahl., Galium bulgaricum Vel.,<br />
Haplophyllum thesioi<strong>de</strong>s G. Don., Hedysarum<br />
tauricum Pallas ex Willd., Jurinea le<strong>de</strong>borii Bunge.,<br />
Pastinaca umbrosa Stev. ex DC.<br />
2. Material and Methods<br />
26<br />
Our study of the flora of the Shoumen Plateau<br />
was conducted on the route method in 1998 – 2009.<br />
The names of the taxons are taken from the Flora of<br />
PR Bulgaria, Vol. І – Х [12]. The update of the<br />
taxons is consistent with APG II [19].<br />
The en<strong>de</strong>mites are represented by Asiov et all.<br />
[20].<br />
The relicts are represented by Gruev and<br />
Kuzmanov [21], Peev [22], Boža et all. [23], Peev et<br />
all. [24].<br />
The conservation statute is recognized using the<br />
following documents: Council Directive 92/43/EEC<br />
of the European Community to protect natural<br />
habitats and of wild fauna and flora [3], Convention<br />
on International Tra<strong>de</strong> in Endangered Species of<br />
Wild Fauna and Flora (CITES) [25], Red book of PR<br />
Bulgaria [16], IUCN Red List for Bulgaria [26],<br />
Biological Diversity Act [17], Or<strong>de</strong>r for special<br />
arrangements for the conservation and use of<br />
medicinal plants [27].<br />
3. Results and Discussion<br />
The analysis of the received data leads to the<br />
following results and conclusions: Two species,<br />
Anacamptis pyramidalis C. Rich. and<br />
Himantoglossum hircinum (L.) Spreng., of the 16<br />
protected and listed as endangered species in the<br />
forest <strong>de</strong>velopment project of the Shumen Forestry<br />
do not fall into any category protected by the Red<br />
Book of PR Bulgaria. They are listed as “rare” in the<br />
Red Book of the district Shumen. Furthermore, in the<br />
Red List of the Bulgarian vascular plants, they are<br />
given similar status – “ vulnerable”. Three of the<br />
species: Atropa belladonna L., Castanea sativa Mill.<br />
and Paeonia tenuifolia L. we did not find on the<br />
territory of the plateau. Himantoglossum hircinum<br />
(L.) Spreng. is incorrectly recor<strong>de</strong>d as located in<br />
Bulgaria and should be replaced with the correct<br />
species name, Himantoglossum caprinum (Bieb.) C.<br />
Koch. The name Celtis caucasica Willd. is obsolete,<br />
now replaced by Celtis glabrata Steven.<br />
As a result of several years of observations, we<br />
found that populations of the following species have<br />
increased: Anacamptis pyramidalis C. Rich.,<br />
Cyclamen coum Mill., Galanthus nivalis L.,<br />
Himantoglossum caprinum (Bieb.) C. Koch., Lilium<br />
martagon L. and Ruta graveolens L. Therefore, they<br />
are not really endangered anymore.
Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
From the 18 protected species inclu<strong>de</strong>d in the<br />
Management Plan of National Park Shumensko<br />
Plateau, 2 species, Colchicum davidovii Stefanov and<br />
Veronica spicata L., listed as endangered, have not<br />
been confirmed by us as existing on the plateau. We<br />
think that Colchicum davidovii Stefanov has<br />
disappeared from the flora of the plateau.<br />
Four species listed as rare or “rare” species, we<br />
did not found on the plateau: Anthemis rumelica<br />
(Velen.) Stoj. et Acht., Fritillaria graeca Boiss. &<br />
Spruner, Galium bulgaricum Vel., Hedysarum<br />
tauricum Pallas ex Willd.<br />
The new data for the conservation statute of the<br />
species, established by us within the realm of the<br />
Shumensko Plateau, shows the following:<br />
The total number of species with conservation<br />
statute is 80 (Figure 1). This is a 10.18% from the<br />
total number of species found on the Shumensko<br />
Plateau. We found the following species:<br />
1. Aegilops geniculata Roth<br />
2. Aesculus hippocastanum L.<br />
3. Althaea officinalis L.<br />
4. Anacamptis pyramidalis C. Rich.<br />
5. Anemone sylvestris L.<br />
6. Anthemis regis-borisii Stoj. et Acht.<br />
7. Artemisia pe<strong>de</strong>montana Balb.<br />
8. Asarum europaeum L.<br />
9. Asparagus tenuifolius Lam.<br />
10. Asparagus verticillatus L.<br />
11. Asplenium trichomanes L.<br />
12. Berberis vulgaris L.<br />
13. Betonica officinalis L.<br />
14. Bupleurum affine Sadl.<br />
15. Bupleurum apiculatum Friv.<br />
16. Bupleurum praealtum L.<br />
17. Bupleurum rotundifolium L.<br />
18. Campanula euxina (Vel.) Ancev<br />
19. Carlina acanthifolia All.<br />
20. Celtis glabrata Steven<br />
21. Centaurea marshalliana Spreng.<br />
22. Cephalanthera damasonium (Mill.) Druce<br />
23. Cephalanthera longifolia (L.) Fritsch<br />
24. Cephalanthera rubra (L.) Rich.<br />
25. Cercis siliquastrum L.<br />
26. Convallaria majalis L.<br />
27. Crocus flavus West.<br />
28. Crocus pallasii Bieb.<br />
29. Cyclamen coum Mill.<br />
30. Dactylorhiza saccifera (Brongn.) Soo<br />
27<br />
31. Dryopteris filix-mas (L.) Schott<br />
32. Echinops sphaerocephalos L.<br />
33. Epipactis helleborine (L.) Crantz<br />
34. Epipactis microphylla (Ehrh.) Sw.<br />
35. Epipactis purpurata Smith<br />
36. Erodium hoefftianum C. A. Mey.<br />
37. Fibigia clypeata (L.) Medic.<br />
38. Fritillaria pontica Wahl.<br />
39. Galanthus elwesii Hook. fil.<br />
40. Galanthus nivalis L.<br />
41. Galium odoratum (L.) Scop.<br />
42. Galium rubioi<strong>de</strong>s L.<br />
43. Gypsophila paniculata L.<br />
44. Haplophyllum thesioi<strong>de</strong>s G. Don.<br />
45. Helichrysum arenarium (L.) Mornh.<br />
46. Himantoglossum caprinum (Bieb.) C. Koch<br />
47. Juniperus sabina L.<br />
48. Jurinea le<strong>de</strong>bourii Bunge<br />
49. Lilium martagon L.<br />
50. Limodorum abortivum (L.) Sw.<br />
51. Listera ovata (L.) R. Br.<br />
52. Neottia nidus-avis (L.) Rich.<br />
53. Ophrys apifera Huds.<br />
54. Ophrys cornuta Stev.<br />
55. Ophrys mammosa Desf.<br />
56. Orchis morio L.<br />
57. Orchis purpurea Huds.<br />
58. Orchis simia Lam.<br />
59. Orchis tri<strong>de</strong>ntata Scop.<br />
60. Pastinaca umbrosa Stev. et DC.<br />
61. Phyllitis scolopendrium (L.) Newm.<br />
62. Platanthera chlorantha (Cust.) Rchb.<br />
63. Polygonatum odoratum (Mill.) Druce<br />
64. Polystichum aculeatum (L.) Roth<br />
65. Primula veris L.<br />
66. Pulmonaria mollis Horn.<br />
67. Ruscus aculeatus L.<br />
68. Ruscus hypoglossum L.<br />
69. Ruta graveolens L.<br />
70. Salix caprea L.<br />
71. Scilla bifolia L.<br />
72. Sedum acre L.<br />
73. Sternbergia colchiciflora Waldst. et Kit.<br />
74. Stipa capillata L.<br />
75. Stipa pulcherrima C. Koch<br />
76. Stipa tirsa Stev.<br />
77. Taxus baccata L.<br />
78. Tilia rubra DC.<br />
79. V<strong>ale</strong>riana officinalis L.
The plants with protection statute, en<strong>de</strong>mites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
80. Vicia pisiformis L.<br />
Two species are inclu<strong>de</strong>d in Application II of<br />
Directive 92/43/ЕЕС: Cyclamen coum Mill. and<br />
Himantoglossum caprinum (Bieb.) C. Koch.<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
2<br />
24<br />
32<br />
21<br />
51<br />
1<br />
12<br />
6<br />
29<br />
42<br />
Directive 92/43/ЕЕС<br />
CITES<br />
IUCN Red List<br />
Red book<br />
BDA<br />
Herbs prohibited from collecting<br />
Herbs in the restrictive regime<br />
En<strong>de</strong>mites<br />
Relicts<br />
Fig. 1. Proportion of species with<br />
conservation status, en<strong>de</strong>mites and relicts<br />
In the Convention on International Tra<strong>de</strong> in<br />
Endangered Species of Wild Fauna and Flora<br />
(CITES) are inclu<strong>de</strong>d 24 species: Anacamptis<br />
pyramidalis C. Rich., Cephalanthera damasonium<br />
(Mill.) Druce, Cephalanthera longifolia (L.) Fritsch,<br />
Cephalanthera rubra (L.) Rich., Cyclamen coum<br />
Mill., Dactylorhiza saccifera (Brongn.) Soo,<br />
Epipactis helleborine (L.) Crantz, Epipactis<br />
microphylla (Ehrh.) Sw., Epipactis purpurata Smith,<br />
Galanthus elwesii Hook. fil., Galanthus nivalis L.,<br />
Himantoglossum caprinum (Bieb.) C. Koch,<br />
Limodorum abortivum (L.) Sw., Listera ovata (L.) R.<br />
Br., Neottia nidus-avis (L.) Rich., Ophrys apifera<br />
Huds., Ophrys cornuta Stev., Ophrys mammosa<br />
Desf., Orchis morio L., Orchis purpurea Huds.,<br />
Orchis simia Lam., Orchis tri<strong>de</strong>ntata Scop.,<br />
Platanthera chlorantha (Cust.) Rchb., Sternbergia<br />
colchiciflora Waldst. et Kit.<br />
The IUCN Red List for Bulgaria are inclu<strong>de</strong>d 32<br />
species. In category „threatened” are inclu<strong>de</strong>d 13<br />
species: Aesculus hippocastanum L., Anthemis regisborisii<br />
Stoj. et Acht., Artemisia pe<strong>de</strong>montana Balb.,<br />
Campanula euxina (Vel.) Ancev, Celtis glabrata<br />
Steven, Epipactis purpurata Smith, Galanthus elwesii<br />
Hook. fil., Galanthus nivalis L., Juniperus sabina L.,<br />
Jurinea le<strong>de</strong>bourii Bunge, Ophrys apifera Huds.,<br />
Ruta graveolens L., Taxus baccata L. In category<br />
„vulnerable” are inclu<strong>de</strong>d 9 species: Anacamptis<br />
pyramidalis C. Rich., Epipactis microphylla (Ehrh.)<br />
Sw., Fibigia clypeata (L.) Medic., Haplophyllum<br />
28<br />
thesioi<strong>de</strong>s G. Don., Himantoglossum caprinum<br />
(Bieb.) C. Koch, Limodorum abortivum (L.) Sw.,<br />
Ophrys cornuta Stev., Ophrys mammosa Desf.,<br />
Pastinaca umbrosa Stev. et DC. In category „nearly<br />
threatened” 5 species: Anemone sylvestris L., Cercis<br />
siliquastrum L., Erodium hoefftianum C. A. Mey.,<br />
Galium rubioi<strong>de</strong>s L., Vicia pisiformis L. In category<br />
„least concern” are inclu<strong>de</strong>d 5 species: Aegilops<br />
geniculata Roth, Cyclamen coum Mill., Fritillaria<br />
pontica Wahl., Pulmonaria mollis Horn., Tilia rubra<br />
DC.<br />
In the Red book for PR Bulgaria are inclu<strong>de</strong>d<br />
total of 21 species. In the category „endangered” are<br />
inclu<strong>de</strong>d 7 species: Aesculus hippocastanum L.,<br />
Anemone sylvestris L., Artemisia pe<strong>de</strong>montana<br />
Balb., Galanthus nivalis L., Galium rubioi<strong>de</strong>s L.,<br />
Ruta graveolens L., Taxus baccata L. In category<br />
„rare” are inclu<strong>de</strong>d 14 species: Anthemis regisborisii<br />
Stoj. et Acht., Celtis glabrata Steven, Cercis<br />
siliquastrum L., Cyclamen coum Mill., Erodium<br />
hoefftianum C. A. Mey., Fibigia clypeata (L.)<br />
Medic., Fritillaria pontica Wahl., Haplophyllum<br />
thesioi<strong>de</strong>s G. Don., Juniperus sabina L., Jurinea<br />
le<strong>de</strong>bourii Bunge, Limodorum abortivum (L.) Sw.,<br />
Pastinaca umbrosa Stev. et DC., Tilia rubra DC.,<br />
Vicia pisiformis L.<br />
In the Biological Diversity Act are inclu<strong>de</strong>d total<br />
of 51 species. In the category „protected”<br />
(Application 3) are inclu<strong>de</strong>d 23 species: Aesculus<br />
hippocastanum L., Anacamptis pyramidalis C. Rich.,<br />
Anemone sylvestris L., Anthemis regis-borisii Stoj. et<br />
Acht., Artemisia pe<strong>de</strong>montana Balb., Campanula<br />
euxina (Vel.) Ancev, Centaurea marshalliana<br />
Spreng., Cyclamen coum Mill., Epipactis purpurata<br />
Smith, Fritillaria pontica Wahl., Galanthus elwesii<br />
Hook. fil., Galanthus nivalis L., Galium rubioi<strong>de</strong>s<br />
L., Haplophyllum thesioi<strong>de</strong>s G. Don.,<br />
Himantoglossum caprinum (Bieb.) C. Koch,<br />
Juniperus sabina L., Jurinea le<strong>de</strong>bourii Bunge,<br />
Limodorum abortivum (L.) Sw., Ophrys apifera<br />
Huds., Ophrys cornuta Stev., Ophrys mammosa<br />
Desf., Ruta graveolens L., Taxus baccata L.<br />
In the category “un<strong>de</strong>r protection and un<strong>de</strong>r<br />
controlled use” (Application 4) are 28 species:<br />
Asparagus tenuifolius Lam., Asparagus verticillatus<br />
L., Bupleurum affine Sadl., Bupleurum apiculatum<br />
Friv., Bupleurum praealtum L., Bupleurum<br />
rotundifolium L., Crocus flavus West., Crocus<br />
pallasii Bieb., Dactylorhiza saccifera (Brongn.) Soo,
Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
Dryopteris filix-mas (L.) Schott, Echinops<br />
sphaerocephalos L., Gypsophila paniculata L.,<br />
Helichrysum arenarium (L.) Mornh., Lilium<br />
martagon L., Orchis morio L., Orchis purpurea<br />
Huds., Orchis simia Lam., Orchis tri<strong>de</strong>ntata Scop.,<br />
Polygonatum odoratum (Mill.) Druce, Polystichum<br />
aculeatum (L.) Roth, Primula veris L., Ruscus<br />
aculeatus L., Ruscus hypoglossum L., Salix caprea<br />
L., Scilla bifolia L., Stipa capillata L., Stipa<br />
pulcherrima C. Koch, Stipa tirsa Stev.<br />
Prohibited is the collecting ofherbs from the<br />
natural habitats of 12 species: Althaea officinalis L.,<br />
Asarum europaeum L., Asplenium trichomanes L.,<br />
Convallaria majalis L., Helichrysum arenarium (L.)<br />
Mornh., Orchis morio L., Orchis purpurea Huds.,<br />
Orchis simia Lam., Orchis tri<strong>de</strong>ntata Scop., Phyllitis<br />
scolopendrium (L.) Newm., Ruscus aculeatus L.,<br />
V<strong>ale</strong>riana officinalis L.<br />
Un<strong>de</strong>r a controlled use are 6 species: Berberis<br />
vulgaris L., Betonica officinalis L., Carlina<br />
acanthifolia All., Galium odoratum (L.) Scop.,<br />
Primula veris L., Sedum acre L.<br />
En<strong>de</strong>mic species (Figure 1) are relatively well<br />
represented – 29 species (3.69% of all species on the<br />
plateau). Their number is close to the nation-wi<strong>de</strong><br />
average – 4.86% [24]. This group inclu<strong>de</strong>s 17 Balkan<br />
suben<strong>de</strong>mites: Campanula grossekii Heuff.,<br />
Campanula lingulata W. et K., Carduus candicans<br />
Waldst. et Kit., Chaerophyllum byzantinum Boiss.,<br />
Doronicum orient<strong>ale</strong> Hoffm., Galium heldreichii<br />
Hal., Galium pasch<strong>ale</strong> Forsskal, Galium<br />
pseudoaristatum Schur., Ophrys cornuta Stev.,<br />
Pseudolysimachion barrelieri (Schott ex Roem. et<br />
Schult.) Holub, Salvia amplexicaulis Lam., Senecio<br />
papposus (Reichenb.) Less., Stachys obliqua Waldst.<br />
et Kit., Symphytum ottomanum Friv., Syringa vulgaris<br />
L., Thesium simplex Vel., Verbascum lychnitis L. The<br />
Balkan en<strong>de</strong>mites are 9 species: Achillea clypeolata<br />
Sibth. et Sm., Aesculus hippocastanum L., Bupleurum<br />
apiculatum Friv., Inula aschersoniana Janka, Knautia<br />
macedonica Griseb., Koeleria simonkaii Adam.,<br />
Onosma thracica Vel., Salvia ringens Sibth. et Sm.,<br />
Sesleria latifolia (Adam.) Deg. The Bulgarian<br />
en<strong>de</strong>mites are 3 species: Anthemis regis-borisii Stoj.<br />
et Acht., Campanula euxina (Vel.) Ancev, Myosotis<br />
aspera Vel.<br />
Data for the relict species on the area of the<br />
plateau was first published by Zahariev and<br />
Radoslavova [14]. The flora of the plateau inclu<strong>de</strong>d<br />
29<br />
significant number of relict species – 42 (Figure 1).<br />
They account for 5.34% of the total species. The<br />
majority of them, 39 species, are Tertiary relicts:<br />
Abies alba Mil., Acer campestre L., Acer hyrcanum<br />
Fisch. et C. A. Meyer, Acer pseudoplatanus L., Acer<br />
tataricum L., Aesculus hippocastanum L., Betula<br />
pendula Roth, Carpinus betulus L., Carpinus<br />
orientalis Mill., Celtis glabrata Steven, Cercis<br />
siliquastrum L., Clematis vitalba L., Corylus<br />
avellana L., Cotinus coggygria Scop., Cyclamen<br />
coum Mill., Fraxinus excelsior L., Fraxinus ornus<br />
L., He<strong>de</strong>ra helix L., Juniperus communis L.,<br />
Lathyrus aureus (Stev.) Brandza, Pastinaca umbrosa<br />
Stev. et DС., Phragmites australis (Cav.) Steud.,<br />
Picea abies (L.) Karsten, Pinus nigra Arn., Populus<br />
alba L., Populus nigra L., Populus tremula L.,<br />
Pteridium aquilinum (L.) Kuhn., Quercus cerris L.,<br />
Quercus d<strong>ale</strong>champii Ten., Ruscus aculeatus L.,<br />
Ruscus hypoglossum L., Salix alba L., Salix caprea<br />
L., Taxus baccata L., Ulmus laevis Pall., Ulmus<br />
minor Mill., Viburnum lantana L., Viscum album L.<br />
They were wi<strong>de</strong>spread during the Tertiary, but their<br />
habitats today are much smaller.<br />
The second group are quaternary relicts. They<br />
have become part of our flora as a result of glaciation<br />
during the Quaternary. Therefore, they are<br />
consi<strong>de</strong>red glacial relicts. On the plateau, there are<br />
two such species: Limodorum abortivum (L.) Sw.<br />
and Galanthus nivalis L. From the third group, the<br />
postglacial steppe relict, only one species is found:<br />
Sternbergia colchiciflora Waldst. et Kit.<br />
The species with highest conservation value, i.e.<br />
those that fall into the categories of being<br />
endangered and vulnerable, are 24 in number.<br />
With the highest conservation value is Cyclamen<br />
coum Mill., which is inclu<strong>de</strong>d in 6 different lists of<br />
endangered species: Directive 92/43/ЕЕС, CITES,<br />
IUCN Red List, Red book, BDA, Tertiary relicts.<br />
Second comes the group of the species<br />
Galanthus nivalis L. and Limodorum abortivum (L.)<br />
Sw. They appear in 5 different lists: CITES, IUCN<br />
Red List, Red book, BDA, quaternary relicts. This<br />
also applies to Aesculus hippocastanum L., which is<br />
inclu<strong>de</strong>d in the following lists: IUCN Red List, Red<br />
book, BDA, Balkan en<strong>de</strong>mites, Tertiary relicts.<br />
The third group of species that is listed in 4 lists<br />
is Himantoglossum caprinum (Bieb.) C. Koch.<br />
(Directive 92/43/ЕЕС, CITES, IUCN Red List, ЗБР),<br />
Anthemis regis-borisii Stoj. et Acht. (IUCN Red List,
The plants with protection statute, en<strong>de</strong>mites and relicts.../ Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
Red book, BDA, Bulgarian en<strong>de</strong>mites), Ophrys<br />
cornuta Stev. (CITES, IUCN Red List, BDA, Balkan<br />
suben<strong>de</strong>mites), Taxus baccata L. (IUCN Red List,<br />
Red book, BDA, Tertiary relicts).<br />
The largest is the group of species that appear in<br />
the following lists:<br />
• CITES, IUCN Red List, BDA – Anacamptis<br />
pyramidalis C. Rich., Epipactis purpurata<br />
Smith, Galanthus elwesii Hook. fil., Ophrys<br />
apifera Huds., Ophrys mammosa Desf.;<br />
• IUCN Red List, Red book, BDA – Anemone<br />
sylvestris L., Fritillaria pontica Wahl., Galium<br />
rubioi<strong>de</strong>s L., Haplophyllum thesioi<strong>de</strong>s G.<br />
Don., Juniperus sabina L., Jurinea le<strong>de</strong>bourii<br />
Bunge, Ruta graveolens L.;<br />
• IUCN Red List, Red book, Tertiary relicts –<br />
Celtis glabrata Steven, Cercis siliquastrum L.,<br />
Pastinaca umbrosa Stev. et DC.;<br />
• IUCN Red List, BDA, Bulgarian en<strong>de</strong>mites –<br />
Campanula euxina (Vel.) Ancev.<br />
4. Conclusions<br />
The total number of species with conservation<br />
statute that we found on the Shoumen plateau is 80<br />
(10.18% of all species on the plateau). It is<br />
significantly larger than the data published by other<br />
authors. In our study, we use more recent documents<br />
on nature conservation. They total 6 in comparison to<br />
3 or 4 in previous publications. The species that we<br />
<strong>de</strong>scribed generally appear in 12 lists of endangered<br />
species.<br />
The en<strong>de</strong>mic species that we found on the plateau<br />
and <strong>de</strong>scribed are 29 species (3.69% of the total<br />
number of species). They inclu<strong>de</strong> 17 Balkan<br />
suben<strong>de</strong>mites, 9 Balkan en<strong>de</strong>mites and 3 Bulgarian<br />
en<strong>de</strong>mites.<br />
The flora of the plateau inclu<strong>de</strong>s significant<br />
number of relict species – 42 (5.34% of the total<br />
number of species). The majority of them are Tertiary<br />
relicts: 39 species, 2 are quaternary relicts and 1 is<br />
postglacial steppe relict.<br />
The largest number of species of conservation<br />
statute confirms the importance of the Shoumen<br />
Plateau as a protected site, preserving the wellbeing<br />
of nature in the future.<br />
30<br />
5. References<br />
[1] Protected Areas Act, State Gazette number 133<br />
from 11 November 1998, Amen<strong>de</strong>d in State<br />
Gazette number 98 from 12 November 1999,...,<br />
Amen<strong>de</strong>d in State Gazette number 19 from 13<br />
March 2009.<br />
[2] ANDREEV, N., 1992. Botanical characteristics<br />
of National Park Shumensko Plateau, in<br />
National Park Shumensko Plateau. Technical<br />
Project Green Construction, Agrolesproject, pp.<br />
17–62.<br />
[3] Council Directive 92/43/EEC of the European<br />
Community to protect natural habitats and of<br />
wild fauna and flora.<br />
[4] STANEV, S., 2001. Little known names from<br />
Bulgarian botany, Pensoft, Sofia – Moscow,<br />
202 pp.<br />
[5] VELENOVSKY, J., 1891. Flora Bulgarica,<br />
Praga, 676 рp.<br />
[6] VELENOVSKY, J., 1898. Flora Bulgarica,<br />
Supplementum I, Praga, 420 рp.<br />
[7] DAVIDOV, B., 1904. Contribution to study the<br />
flora of the district of Shumen, Sbornik ot<br />
narodni umotvoreniya, ХХ (II): 1–54.<br />
[8] STOIANOV, N., Stefanov B., 1924-1925. Flora<br />
of Bulgaria, Vol. I-II, Sofia, pp. 1367.<br />
[9] STOIANOV, N., Stefanov B., 1932-1933. Flora<br />
of Bulgaria, Vol. I-II, Sofia.<br />
[10] STOIANOV, N., Stefanov B., 1947-1948. Flora<br />
of Bulgaria, Vol. I-II, Sofia, pp. 1361.<br />
[11] STOIANOV, N., Stefanov B., Kitanov, B.,<br />
1966-1967. Flora of Bulgaria, Vol. I-II, Nauka i<br />
izkustvo, Sofia, pp. 1325.<br />
[12] Flora of PR Bulgaria, Vol. І-Х, 1963-1995,<br />
Publishing House of BAS, Sofia.<br />
[13] RADOSLAVOVA, Е., 2002. The Orchids of the<br />
Shumensko Plateau, Snejanka Petkova – AR,<br />
Shumen, pp. 48.<br />
[14] ZAHARIEV, D., Radoslavova, E., 2010. The<br />
Plants of the Shumensko Plateau, Himera,<br />
Shumen, pp. 597.<br />
[15] Forest <strong>de</strong>velopment project of the Shumen State<br />
Forestry, district Shumen, Vol. I, 2002,<br />
Anemone Ltd., Sofia, pp. 148.<br />
[16] Red book of PR Bulgaria, Vol. 1, Plants, 1984,<br />
Publishing House of BAS, Sofia, 447 pp.<br />
[17] Biological Diversity Act, State Gazette number<br />
77 from 9 august 2002, pp. 9–42. Amen<strong>de</strong>d in
Dimcho Zahariev, Elka Radoslavova / Ovidius University Annals of Biology-Ecology 14: 25-31 (2010)<br />
State Gazette number 94 from 16 November<br />
2007.<br />
[18] BESHKOV, V. et all.. (eds.), 1994. The Red<br />
book of the district Shumen, Slavcho Nikolov &<br />
co, Shumen, pp. 199.<br />
[19] CHASE, M. (corresponding author), 2003. An<br />
update of the Angiosperm Phylogeny Group<br />
classification for the or<strong>de</strong>rs and families of<br />
flowering plants: APG II, The Linnean Society<br />
of London, Botanical Journal of the Linnean<br />
Society, 141: 399–436.<br />
[20] ASIOV B., Petrova A., Dimitrov D., Vasilev R.,<br />
2006. Conspectus of the Bulgarian vascular<br />
flora. Distribution maps and floristic elements,<br />
Bulgarian Biodiversity Foundation, Sofia, 452<br />
pp.<br />
[21] GRUEV, B., Kuzmanov B., 1994. General<br />
biogeography, University Press St. Kliment<br />
Ohridski, Sofia, 498 pp.<br />
[22] PEEV, D., 2001. National park Rila.<br />
Management plan 2001, 2010. Adopted by<br />
Resolution №522 of Council of Ministers on<br />
04.07.2001, Sofia, 338 pp.<br />
[23] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić<br />
D., 2005. Flora “Rimskog šanca” (Vojvodina,<br />
Srbija), 8th Symposium on the flora of<br />
Southeastern Serbia and Neighbouring Regions,<br />
Niš, 20-24.06.2005, Abstracts, рр. 55.<br />
[24] PEEV, D., Kozuharov S., Anchev M., Petrova<br />
A., Ivanova D., Tzoneva S., 1998. Biodiversity<br />
of Vascular Plants in Bulgaria, In: Curt Meine<br />
(ed.), Bulgaria's Biological Diversity:<br />
Conservation Status and Needs Assessment,<br />
Volumes I and II, Washington, D.C.,<br />
Biodiversity Support Program, pp. 55–88.<br />
[25] Convention on International Tra<strong>de</strong> in<br />
Endangered Species of Wild Fauna and Flora,<br />
State Gazette number 6 from 21 Januari 1992.<br />
[26] PETROVA А., Vladimirov V. (eds.), 2009. Red<br />
List of Bulgarian vascular plants, Phytologia<br />
Balcanica 15 (1): 63–94.<br />
[27] Or<strong>de</strong>r number RD-72 from 3 februari 2006 for<br />
special arrangements for the conservation and use<br />
of medicinal plants, State Gazette number 16<br />
from 21 Februari 2006.<br />
31
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
A CHARACTERISTIC OF MODEL HABITATS IN SOUTH DOBRUDJA<br />
Dimcho ZAHARIEV<br />
University of Shumen Bishop Konstantin Preslavski, Faculty of Nature Sciences,<br />
115 Universitetska Str., 9712, Shumen, Bulgaria, dimtchoz@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: Five natural habitats and five artificial habitats (forest shelter belts) are investigated in South<br />
Dobrudja. Most taxonomical diversity and most protected species from natural habitats are established in Western<br />
Pontic Paeonian steppes near to Bejanovo village. In the forest shelter belts is typical less taxonomical diversity,<br />
less protected species and more anthropophytes, which due to strong anthropogenically influence. The families<br />
with most of genera and species are: Asteraceae, Рoaceae, Rosaceae and Lamiaceae. The biggest groups from<br />
biological types are perennial herbaceous plants and annual herbaceous plants. The floristic elements are<br />
presented mainly from circumboreal, European and Mediterranean elements. The mainly reasons about high<br />
number of anthropophytes are intensive fragmentation of the natural habitats, all round from agricultural areas – a<br />
source of anthropophytes, and their accessibility for peoples and domestic animals.<br />
Keywords: Dobrudja, habitats, taxonomical diversity, biological types, floristic elements, en<strong>de</strong>mites, relict<br />
species, protected species, anthropophytes.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Dobrudja is historical and geographical area<br />
between the lower reaches of the Danube and Black<br />
Sea. The area is 23 000 km 2 . It is divi<strong>de</strong>d into two<br />
parts – North and South Dobrudja.<br />
North Dobrudja is located in Southeastern<br />
Romania. Its area covers about 2/3 of the territory,<br />
amounting to 15 435 km².<br />
South Dobrudja is located in Northeastern<br />
Bulgaria. The area is 7 565 km². The Bulgarian part<br />
of Dobrudja is divi<strong>de</strong>d by the virtual line between<br />
Stojer village and Rosica village into two parts –<br />
eastern and western. South Dobrudja is located in 3<br />
administrative areas – Varnenska (municipality<br />
Aksakovo), Dobrichka (all municipalities) and<br />
Silistrenska (municipality Kainardja).<br />
The climate is temperate. It is characterized by<br />
warm summers and cold winters, high annual<br />
amplitu<strong>de</strong> of air temperature, spring–summer<br />
minimum and winter maximum of rainfall, the snow<br />
cover is relatively stable. The average temperatures in<br />
January are between 0°С and –1.5°С. In the summer<br />
dominated tropical and subtropical air masses and the<br />
average temperature in July is 22-24°С. The spring<br />
and the autumn are approximately the same<br />
temperatures. April was warmer in October. The<br />
rainfalls are with maximum in May–June and with<br />
minimum in February–March. The annual amount of<br />
precipitation is 520 to 650 mm. About 10% of the<br />
total amount of precipitation is snow [1].<br />
In South Dobrudja dominated haplic Chernozems.<br />
Small areas are covered with kastanic, calcaric<br />
or gleyc Chernozems. On the coast of the Black Sea<br />
and the rivers are distributed rendzic Leptosols and<br />
Nitisols. Along the Danube are distributed calcaric<br />
Fluvisols, Histosols and Gleysols. Unique to the<br />
region are the small in area Vertisols [2].<br />
In terms of its flora, South Dobrudja belongs to<br />
the region of Northeastern Bulgaria. On its territory<br />
are <strong>de</strong>scribed 1 508 species, which are referred to 496<br />
genera and 144 families. Natural vegetation was<br />
composed of forest steppes, which inclu<strong>de</strong> large<br />
forest complexes and grasslands. Original natural<br />
vegetation of the Dobrudja was <strong>de</strong>stroyed in a large<br />
part, due to intensive human activities [3]. Today on<br />
the territory of Southern Dobrudja occur 41 different<br />
plant communities – primary and secondary [4]. 33<br />
habitats are <strong>de</strong>scribed according to Council Directive<br />
92/43/EEC of the European Community to protect<br />
ISSN-1453-1267 © 2010 Ovidius University Press
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
natural habitats and of wild fauna and flora [5, 6, 7, 8,<br />
9, 10, 11, 12, 13, 14].<br />
2. Material and Methods<br />
The field studies were conducted on the route<br />
method in 2008 – 2009. Subject of research are a<br />
total of 10 different habitats – 5 natural and 5<br />
artificial. The natural habitats are <strong>de</strong>fined by<br />
Kavrakova et all. [15]. Each habitat is characterized<br />
as follows: average altitu<strong>de</strong>, exposure, slope, area,<br />
soil type and subtype, base rock, cover of tree, shrub<br />
and herbaceous vegetation, number of established<br />
species, genera and families, cover of each species,<br />
distribution in biological type, floristic elements,<br />
en<strong>de</strong>mites, suben<strong>de</strong>mites and relict species, species<br />
with conservation status, anthropophytes,<br />
anthropogenic influence. The average altitu<strong>de</strong>,<br />
exposure, slope and area are <strong>de</strong>fined with map at a<br />
sc<strong>ale</strong> 1:50 000. The soil types and subtypes are<br />
presented by Ninov [2]. The taxons and the biological<br />
type are <strong>de</strong>fined by I<strong>de</strong>ntifier of the vascular plants in<br />
Bulgatia [16], Flora of PR Bulgaria, Vol. І – Х [17].<br />
The update of the taxons is consistent with APG II<br />
[18] and Petrova et all. [19]. The cover of each<br />
species is presented by Braun-Blanquet [20]. The<br />
following symbols are used: r – cover less than 5%,<br />
one individual; + – cover less than 5%, 2-5<br />
individuals; 1 – cover less than 5%, 6-50 individuals;<br />
2m – cover less than 5%, more than 50 individuals;<br />
2a – cover 5-12.5%; 2b – cover 12.5-25%; 3 – cover<br />
25-50%; 4 – cover 50-75%; 5 – cover 75-100%. The<br />
following symbols are used [16] for biological types:<br />
t (from English tree), sh (from English shrub), p<br />
(from English perennial), а (from English annual).<br />
The floristic elements, en<strong>de</strong>mites and suben<strong>de</strong>mites<br />
are presented by Asiov et all. [21]. The relicts are<br />
presented by Gruev and Kuzmanov [22], Peev [23],<br />
Boža et all. [24], Peev et all. [25]. The conservation<br />
status is presented using the following documents:<br />
Council Directive 92/43/EEC of the European<br />
Community to protect natural habitats and of wild<br />
fauna and flora [26], Berne Convention [27],<br />
Convention on International Tra<strong>de</strong><br />
in Endangered Species of Wild Fauna and Flora<br />
(CITES) [28], Red book of PR Bulgaria [29], IUCN<br />
Red List for Bulgaria [30], Biological Diversity Act<br />
[27], Or<strong>de</strong>r for special arrangements for the<br />
conservation and use of medicinal plants [31]. The<br />
34<br />
anthropophytes are presented by Stefanov and<br />
Kitanov [32]. is recor<strong>de</strong>d by the system of effects<br />
used in the assessment of an object from the network<br />
of protected areas Nature 2000.<br />
3. Results and Discussion<br />
HABITAT 1<br />
A habitat by Nature 2000: Euro-Siberian<br />
steppic woods with Quercus spp.<br />
A habitat by Bon<strong>de</strong>v [4]: Cerris oak (Querceta<br />
cerris) forests.<br />
It is located southwest of Efreitor Bakalovo<br />
village, municipality Krushari. The territory is a part<br />
of Nature 2000 (Protected area “Suha reka”). The<br />
average altitu<strong>de</strong> is 150 m. The exposure is south. The<br />
slope varies in different parts. It is smaller in the<br />
north and higher in southern parts. The area of the<br />
habitat is 6 300 dka. The soil type is Chernozems,<br />
and the soil subtype is haplic Chernozems. The<br />
bedrock is limestone. The cover of the tree vegetation<br />
is 80%, the cover of the shrub vegetation is 10% and<br />
the cover of the herbaceous vegetation is 10%.<br />
In the habitat have been in<strong>de</strong>tified 78 species of<br />
vascular plants from 67 genera and 27 families. The<br />
families with greatest number of genera are as<br />
follows: Asteraceae – 8 (11.94%), Poaceae – 8<br />
(11.94%), Brassicaceae – 5 (7.46%) and Fabaceae –<br />
5 (7.46%). The families with greatest number of<br />
species are as follows: Poaceae – 10 (12.82%),<br />
Rosaceae – 9 (11.54%), Asteraceae – 8 (10.26%),<br />
Brassicaceae – 6 (7.69%), Fabaceae – 5 (6.41%),<br />
Lamiaceae – 5 (6.41%) and Scrophulariaceae – 5<br />
(6.41%). The genera with greatest number of species<br />
are as follows: Veronica – with 4 species (5.13%),<br />
Poa – with 3 species (3.85%) and Potentilla – with 3<br />
species (3.85%).<br />
With the highest percentage of coverage are<br />
Quercus cerris L. (5) and Poa nemoralis L. (2a).<br />
With the lowest percentage of coverage (1) are<br />
Cornus mas L., Carduus nutans L. and Vicia sativa<br />
L. Each of the remaining 73 species has coverage 2m.<br />
The distribution of species in biological type is<br />
as follows: The perennial herbaceous plants (p) are<br />
most – they are 38 species (48.72%). Secondly, are
Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
annual herbaceous plants (a) with 21 species<br />
(26.92%). The next is the transition group of annual<br />
to biennial herbaceous plants (a-b) with 6 species<br />
(7.69%). The trees (t) and the transition group of<br />
shrubs to trees (sh-t) have equal number of species –<br />
4 (5.13%). The biennial herbaceous plants (b) are 3<br />
species (3.85%), and shrubs (sh) are 2 species<br />
(2.56%) only.<br />
The diversity of floristic elements is as follows:<br />
The largest number of species (28) has circumboreal<br />
origin. The next are species with European origin –<br />
they are 25 species. 13 species have Mediterranean<br />
origin. The Pontic type of elements and<br />
cosmopolitans are 5 species each of them. One of the<br />
species is adventive element. One of the species is<br />
Balkan suben<strong>de</strong>mite – Ornithogalum sibthorpii<br />
Greut.<br />
Three species are Tertiary relicts: Carpinus<br />
orientalis Mill., Quercus cerris L. and Ulmus minor<br />
Mill.<br />
Two species with protection statute are<br />
established – Crocus flavus West. and Scilla bifolia<br />
L. They are inclu<strong>de</strong>d in the Biological Diversity Act<br />
in the category „Un<strong>de</strong>r the protection and regulated<br />
use of nature”.<br />
The anthropophytes are 55 species (70.51%).<br />
The large number indicates for increased<br />
anthropogenic impact on the habitat.<br />
The anthropogenic influence consists in the<br />
following: 1. Forestry felling. 2. Artificial<br />
afforestation. 3. Grazing sheep, goats and cows. 4.<br />
Pollution by garbage from the shepherds and farm<br />
workers. 5. Arable land in the vicinity. 6. Improved<br />
access to the habitat by a system of paths and roads.<br />
7. Тourist pavilion with a fireplace. 8. Fountain with<br />
several troughs.<br />
HABITAT 2<br />
A habitat by Bon<strong>de</strong>v [4]: Shrub (Amygd<strong>ale</strong>ta<br />
nanae) and grass (Artemisieta albae, Agropyreta<br />
pectiniformae, Agropyreta brandzae, Brometa riparii<br />
etc.) steppe and xerothermal communities.<br />
It is located south of Karapelit village,<br />
municipality Dobrich. The territory is a part of Nature<br />
2000 (Protected area “Suha reka”). The average<br />
altitu<strong>de</strong> is 160 m. The exposure is in some parts<br />
south, while in others – west. The slope is variable<br />
35<br />
and reaches 30°. The area of the habitat is 400 dka.<br />
The soil type is Chernozems, and the soil subtype is<br />
haplic Chernozems. The bedrock is limestone. The<br />
cover of the shrub vegetation is less<br />
than 5% and the cover of the herbaceous vegetation is<br />
90%. More than 5% of the ground is <strong>de</strong>void of<br />
vegetation cover.<br />
In the habitat have been in<strong>de</strong>tified 83 species of<br />
vascular plants from 70 genera and 24 families. The<br />
families with greatest number of genera are as<br />
follows: Rosaceae – 10 (14.29%), Asteraceae – 7<br />
(10.00%), Lamiaceae – 7 (10.00%), Poaceae – 7<br />
(10.00%) and Apiaceae – 5 (7.14%). The families<br />
with greatest number of species are as follows:<br />
Rosaceae – 10 (12.05%), Lamiaceae – 10 (12.05%),<br />
Asteraceae – 9 (10.84%), Poaceae – 7 (8.43%),<br />
Apiaceae – 5 (6.02%), Caryophillaceae – 5 (6.02%)<br />
and Ranunculaceae – 5 (6.02%). The genera with<br />
greatest number of species are as follows: Euphorbia<br />
and Salvia – with 3 species each of them (3.61%).<br />
Stipa capillata L. (2а) is with the highest<br />
percentage of coverage. With the lowest percentage<br />
of coverage are Robinia pseudoacacia L. (1), Althaea<br />
cannabina L. (1), Prunus mah<strong>ale</strong>b L. (+), Carduus<br />
nutans L. (+), Pyrus pyraster Burgsd. (+), Ligustrum<br />
vulgare L. (r) and Malus sylvestris Mill. (r). From the<br />
neighboring shelter belt immigrated some tree<br />
species. The reason for this is the transference of<br />
fruits and seeds by birds and wind. Each of the<br />
remaining 75 species has coverage 2m.<br />
The distribution of species in biological type is<br />
as follows: The perennial herbaceous plants (p) are<br />
most – they are 48 species (57.83%). Secondly, are<br />
annual herbaceous plants (a) with 18 species<br />
(21.69%). The biennial herbaceous plants (b) and the<br />
trees have 4 species each of them (4.82%). The<br />
shrubs (sh) are 3 species (3.61%). The transition<br />
group of shrubs to trees (sh-t) and the transition group<br />
of annual to perennial herbaceous plants (a-р) have 2<br />
species each of them (2.41%). The transition groups<br />
of annual to biennial herbaceous plants (a-b) and of<br />
biennial to perennial herbaceous plants (b-р) have<br />
one species (1.20%) each of them.<br />
The most species are species with Mediterranean<br />
(23 species), European (22 species) and circumboreal<br />
origin (20 species). The Pontic type of elements are<br />
10 species. The cosmopolitans are 3 species. The<br />
Balkan en<strong>de</strong>mites are 2 species – Achillea clypeolata
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
Sibth. et Sm. and Salvia ringens Sibth. et Sm. One<br />
species is Balkan suben<strong>de</strong>mite – Dianthus pallens<br />
Sm. One species has adventive origin and one species<br />
has Alpine-Mediterranean.<br />
Four species with protection statute are<br />
established: Adonis vernalis L. is inclu<strong>de</strong>d in CITES<br />
and in the Or<strong>de</strong>r for special arrangements for the<br />
conservation and use of medicinal plants in the<br />
category “Collecting herbs is prohibited from the<br />
natural habitats”. Jurinea le<strong>de</strong>bourii Bunge is<br />
inclu<strong>de</strong>d in the IUCN Red List for Bulgaria in the<br />
category “Endangered”, in the Red book for Bulgaria<br />
in the category „Rare” and in the Biological Diversity<br />
Act in the category „Protected”. Two species are<br />
inclu<strong>de</strong>d in the Biological Diversity Act in the<br />
category „Un<strong>de</strong>r the protection and regulated use of<br />
nature”: Bupleurum affine Sadl. and Stipa capillata<br />
L.<br />
The anthropophytes are 53 species (63.86%).<br />
They are an indicator of the extent of human impact<br />
on habitat.<br />
The anthropogenic influence on the habitat due<br />
to the presence of: 1. Improved access to the habitat<br />
by a system roads. 2. Arable land in the vicinity. 3.<br />
The forest shelter belts in the vicinity.<br />
HABITAT 3<br />
A habitat by Bon<strong>de</strong>v [4]: Mesoxerothermal<br />
grass vegetation with a prev<strong>ale</strong>nce of Poa bulbosa L.,<br />
Lolium perenne L., Cynodon dactylon L., partly also<br />
Dichantium ischaemum (L.) Roberty and rarely<br />
Chrysopogon gryllus (L.) Tryn.<br />
It is located between Izvorovo and Krasen<br />
villages, municipality General Toshevo. The average<br />
altitu<strong>de</strong> is 180 m. The exposure is southwest. The<br />
slope is variable and reaches 30°. The area of the<br />
habitat is 2 000 dka. The soil type is Leptosols, and<br />
the soil subtype is rendzic Leptosols. The bedrock is<br />
limestone. The cover of the shrub vegetation is less<br />
than 5% and the cover of the herbaceous vegetation is<br />
80%. More than 15% of the ground is <strong>de</strong>void of<br />
vegetation cover.<br />
In the habitat have been in<strong>de</strong>tified 103 species of<br />
vascular plants from 84 genera and 32 families. The<br />
families with greatest number of genera are as<br />
follows: Asteraceae – 15 (14.56%), Poaceae – 12<br />
(14.29%), Lamiaceae – 10 (11.90%) and<br />
36<br />
Brassicaceae – 5 (5.95%). The families with greatest<br />
number of species are as follows: Poaceae – 14<br />
(13.59%), Lamiaceae – 13 (12.62%), Asteraceae – 12<br />
(11.65%), Brassicaceae – 5 (4.85%), Euphorbiaceae<br />
– 5 (4.85%) and Rubiaceae – 5 (4.85%). The genus<br />
Euphorbia is with greatest number of species – with 5<br />
species (4.85%).<br />
With the highest percentage of coverage are Poa<br />
pratensis L. (2b) and Elymus repens (L.) Gould. (2а).<br />
With the lowest percentage of coverage (1) is<br />
Carduus thoermeri Weinm. Each of the remaining<br />
100 species has coverage 2m.<br />
The distribution of species in biological type is<br />
as follows: The perennial herbaceous plants (p) are<br />
most – they are 53 species (51.46%). Secondly, are<br />
annual herbaceous plants (a) with 32 species<br />
(31.07%). The biennial herbaceous plants (b) are 10<br />
species (9.71%). The transition group of annual to<br />
biennial herbaceous plants (a-b) has 3 species<br />
(2.91%). The transition group of annual to perennial<br />
herbaceous plants (a-р) has 2 species (1.94%). The<br />
trees (t) are 2 species (1.94%) and the shrubs (sh) –<br />
one species (3.61%) only.<br />
The largest number of species (31) has<br />
circumboreal origin. Secondly, are European (27<br />
species) and Mediterranean type of elements (20<br />
species). The species with Pontic origin are 13. The<br />
cosmopolitans are 6 species. Three species are<br />
Balkan suben<strong>de</strong>mites – Ornithogalum sibthorpii<br />
Greut., Verbascum banaticum Schrad. and Carduus<br />
thoermeri Weinm. The adventive species are 2. One<br />
species has Alpine-Carpathian origin.<br />
Three species with protection statute are<br />
established: Artemisia pe<strong>de</strong>montana Balb. is inclu<strong>de</strong>d<br />
in IUCN Red List for Bulgaria in the category<br />
„Endangered”, in the Red book for Bulgaria in the<br />
category „Threatened with extinction” and in the<br />
Biological Diversity Act in the category „Protected”.<br />
Helichrysum arenarium (L.) Mornh. and Stipa<br />
capillata L. are inclu<strong>de</strong>d in the Biological Diversity<br />
Act in the category “Un<strong>de</strong>r the protection and<br />
regulated use of nature”. Helichrysum arenarium (L.)<br />
Mornh. is inclu<strong>de</strong>d in the Or<strong>de</strong>r for special<br />
arrangements for the conservation and use of<br />
medicinal plants in the category “Collecting herbs is<br />
prohibited from the natural habitats”.
Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
The anthropophytes are 80 species (77.67%).<br />
They show a significant anthropogenic impact on the<br />
habitat.<br />
The anthropogenic influence on the habitat due<br />
to the presence of: 1. Improved access to the habitat<br />
by a system roads. 2. Arable land in the vicinity. 3.<br />
Transmission line, passing through the territory. 4.<br />
Grazing sheep and goats. 5. Pollution by garbage<br />
from the two villages. 6. Artificial terracing of slopes.<br />
HABITAT 4<br />
A habitat by Nature 2000: Western Pontic<br />
Paeonian steppes<br />
It is located near Bejanovo village, municipality<br />
General Toshevo. The territory is a part of Nature<br />
2000 (Protected area “Kraimorska Dobrudja”). The<br />
average altitu<strong>de</strong> is 80 m. The exposure is northeast.<br />
The slope is low and reaches 5°. The area of the<br />
habitat is 650 dka. The soil type is Chernozems, and<br />
the soil subtype is calcaric Chernozems. The bedrock<br />
is limestone. The cover of the shrub vegetation is less<br />
than 5% and the cover of the herbaceous vegetation is<br />
70%. More than 25% of the ground is <strong>de</strong>void of<br />
vegetation cover.<br />
In the habitat have been in<strong>de</strong>tified 153 species of<br />
vascular plants from 116 genera and 36 families. It is<br />
the richest of plant species from the natural habitats.<br />
The families with greatest number of genera are as<br />
follows: Asteraceae – 13 (11.21%), Rosaceae – 12<br />
(10.34%), Lamiaceae – 11 (9.48%), Poaceae – 11<br />
(9.48%), Boraginaceae – 6 (5.17%), Brassicaceae – 6<br />
(5.17%), Fabaceae – 6 (5.17%), Apiaceae – 5<br />
(4.31%), Ranunculaceae – 5 (4.31%) and<br />
Scrophulariaceae – 5 (4.31%). The families with<br />
greatest number of species are as follows: Asteraceae<br />
– 18 (11.76%), Rosaceae – 17 (11.11%), Lamiaceae<br />
– 15 (9.80%), Poaceae – 14 (9.15%), Boraginaceae –<br />
8 (5.23%), Caryophyllaceae – 8 (5.23%), Fabaceae –<br />
7 (4.58%), Apiaceae – 6 (3.92%), Brassicaceae – 6<br />
(3.92%), Ranunculaceae – 6 (3.92%), Euphorbiaceae<br />
– 5 (3.27%), Rubiaceae – 5 (3.27%) and<br />
Scrophulariaceae – 5 (3.27%). The genera with<br />
greatest number of species are as follows: Euphorbia<br />
– with 5 species (3.27%), Cerastium, Potentilla,<br />
Prunus, Salvia and Silene – with 3 species each of<br />
them (1.96%).<br />
With the highest percentage of coverage (2b) are<br />
Festuca pseudovina Hack. ex Wiesd., Poa pratensis<br />
37<br />
L. and Stipa capillata L. With the cover 1 are 14<br />
species. With the lowest percentage of coverage are<br />
Malus dasyphylla Borkh. (+) and Cydonia oblonga<br />
Mill. (r). They are most likely carried by birds. Each<br />
of the remaining 134 species has coverage 2m.<br />
The distribution of species in biological type is<br />
as follows: The perennial herbaceous plants (p) are<br />
most – they are 88 species (57.52%). Secondly, are<br />
annual herbaceous plants (a) with 38 species<br />
(24.84%). The next is the transition group of annual<br />
to biennial herbaceous plants (a-b) with 7 species<br />
(4.58%). The trees (t) and the shrubs (sh) are 5<br />
species each of them (3.27%). The biennial<br />
herbaceous plants (b) are 4 species (2.61%). The<br />
transition groups of annual to perennial herbaceous<br />
plants (a-р), of biennial to perennial herbaceous<br />
plants (b-p) and of shrubs to trees (sh-t) have 2<br />
species each of them (1.31%).<br />
The largest number of species (39) has<br />
circumboreal origin. The next are species with<br />
Mediterranean and European type of elements – with<br />
37 species each of them. Thirdly, are the Pontic type<br />
of elements with 21 species. The cosmopolitan are 6<br />
species. Four of the species are Balkan en<strong>de</strong>mites –<br />
Achillea clypeolata Sibth. et Sm., Astragalus<br />
spruneri Boiss., Chamaecytisus jankae (Vel.) Rothm.<br />
and Potentilla emili-popii Nyar. Five of the species<br />
are Balkan suben<strong>de</strong>mites – Carduus thoermeri<br />
Weinm., Centaurea napulifera Roch., Dianthus<br />
pallens Sm., Ornithogalum sibthorpii Greut. and<br />
Thesium simplex Vel. The remaining 3 species have<br />
Alpine-Mediterranean, Oriental-Turanian and<br />
Pannonian-Pontic origin.<br />
Eight species with protection statute are<br />
established: Paeonia tenuifolia L. is inclu<strong>de</strong>d in<br />
Berne Convention, in Directive 92/43/ЕЕС and in the<br />
Biological Diversity Act in the category “Protected”.<br />
Potentilla emili-popii Nyar. is inclu<strong>de</strong>d in Berne<br />
Convention, in Directive 92/43/ЕЕС, in the<br />
Biological Diversity Act in the category „Declaration<br />
of protected areas to protect habitat for species by<br />
Directive 92/43/ЕEC” and in the category<br />
„Protected”. Adonis vernalis L. is inclu<strong>de</strong>d in CITES<br />
and in the Or<strong>de</strong>r for special arrangements for the<br />
conservation and use of medicinal plants in the<br />
category “Collecting herbs is prohibited from the<br />
natural habitats”. Artemisia pe<strong>de</strong>montana Balb. is<br />
inclu<strong>de</strong>d in IUCN Red List for Bulgaria in the
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
category „Endangered”, in the Red book for Bulgaria<br />
in the category „Threatened with extinction” and in<br />
the Biological Diversity Act in the category<br />
„Protected”. Erodium hoefftianum C. A. Mey. is<br />
inclu<strong>de</strong>d in the Red book for Bulgaria in the category<br />
„Rare” and in IUCN Red List for Bulgaria in the<br />
category „Near Threatened”. Pulsatilla montana<br />
(Hoppe) Reichenb., Stipa capillata L. and Stipa<br />
lessingiana Trin. et Rupr. are inclu<strong>de</strong>d in the<br />
Biological Diversity Act in the category “Un<strong>de</strong>r the<br />
protection and regulated use of nature”.<br />
The anthropophytes are 92 species (60.13%),<br />
which indicates a high anthropogenic impact on the<br />
habitat.<br />
The anthropogenic influence on the habitat due<br />
to the presence of: 1. Improved access to the habitat<br />
by a system roads. 2. Arable land in the vicinity. 3.<br />
The forest shelter belts and artificial forest from<br />
Robinia pseudoacacia L. in the vicinity. 4. Grazing<br />
cows. 5. Disposal of soil in the vicinity.<br />
HABITAT 5<br />
A habitat by Nature 2000: Rupicolous<br />
calcareous or basophilic grasslands of the Alysso-<br />
Sedion albi.<br />
It is located between Onogur and Efreitor<br />
Bakalovo villages, municipality Krushari. The<br />
territory is a part of Nature 2000 (Protected area<br />
“Suha reka”). The average altitu<strong>de</strong> is 70 m. The<br />
exposure is south. The slope is variable and reaches<br />
40°. The area of the habitat is 30 dka. The soil type is<br />
Leptosols, and the soil subtype is rendzic Leptosols.<br />
The bedrock is limestone. The cover of the shrub<br />
vegetation is less than 5% and the cover of the<br />
herbaceous vegetation is 30%. More than 65% of the<br />
ground is <strong>de</strong>void of vegetation cover.<br />
In the habitat have been in<strong>de</strong>tified 46 species of<br />
vascular plants from 43 genera and 22 families. It is<br />
the most poor of plant species from the natural<br />
habitats. The families with greatest number of genera<br />
are as follows: Asteraceae – 7 (16.28%), Рoaceae – 5<br />
(11.63%), Lamiaceae and Apiaceae – with 4 species<br />
each of them (9.30%). The families with greatest<br />
number of species are as follows: Asteraceae – 8<br />
(17.39%), Lamiaceae and Poaceae – with 5 species<br />
each of them (10.87%). The genera with greatest<br />
number of species are as follows: Centaurea, Sedum<br />
and Teucrium – with 2 species each of them (4.35%).<br />
38<br />
With the highest percentage of coverage (2а) is<br />
Dichantium ischaemum (L.) Roberty. With the lowest<br />
percentage of coverage (+) are Gleditsia triacanthos<br />
L., Cornus sanguinea L. and Prunus spinosa L. Each<br />
of the remaining 42 species has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 24 species (52.17%). Secondly, are annual<br />
herbaceous plants (a) with 8 species (17.39%). The<br />
shrubs are 6 species (13.04%). The biennial<br />
herbaceous plants (b) are 3 species (6.52%). The<br />
transition group of shrubs to trees (sh-t) has 2 species<br />
(4.35%). The trees (t), the transition groups of annual<br />
to biennial herbaceous plants (а-b) and of annual to<br />
perennial herbaceous plants (a-р) have one species<br />
(2.17%) each of them.<br />
The largest number of species (14) has<br />
Mediterranean origin. Secondly, are circumboreal<br />
type of elements with 11 species. The next are species<br />
with Pontic (9 species) and European origin (8<br />
species). The cosmopolitan are 3 species. One of the<br />
species is adventive element.<br />
Two species with protection statute are<br />
established: Stipa capillata L. is inclu<strong>de</strong>d in the<br />
Biological Diversity Act in the category „Un<strong>de</strong>r the<br />
protection and regulated use of nature”. Sedum acre<br />
L. is inclu<strong>de</strong>d in the Or<strong>de</strong>r for special arrangements<br />
for the conservation and use of medicinal plants in<br />
the category “Un<strong>de</strong>r a restrictive regime”.<br />
The anthropophytes are 32 species (69.57%).<br />
The high rate is due to human activities in adjacent<br />
areas of the habitat.<br />
The anthropogenic influence on the habitat due<br />
to the presence of: 1. Improved access to the habitat<br />
by a system roads. 2. Grazing cows in the bottom of<br />
the slope. 3. Arable land in the vicinity.<br />
HABITAT 6<br />
Forest shelter belt formed by Quercus cerris L.<br />
It is located between General Toshevo and<br />
Liuliakovo village, municipality General Toshevo.<br />
The average altitu<strong>de</strong> is 210 m. The exposure is west.<br />
The shelter belt is oriented in a southwest – northeast<br />
direction. The slope is low and reaches 5°. The area<br />
is 75 dka. The length of the shelter belt is 5 000 m,<br />
and the width – 15 m. The soil type is Chernozems,<br />
and the soil subtype is haplic Chernozems. The<br />
bedrock is limestone. The cover of the tree vegetation
Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
is 80%, the cover of the shrub vegetation is 10% and<br />
the cover of the herbaceous vegetation is 60%.<br />
In the shelter belt have been in<strong>de</strong>tified 49 species<br />
of vascular plants from 44 genera and 21 families.<br />
The families with greatest number of genera are as<br />
follows: Asteraceae and Рoaceae – with 8 species<br />
each of them (18.18%), Rosaceae – 6 (13.64%) and<br />
Lamiaceae – 4 (9.09%). The families with greatest<br />
number of species are as follows: Asteraceae and<br />
Рoaceae – with 8 species each of them (16.33%),<br />
Rosaceae – 6 (12.24%) and Lamiaceae – 4 (8.16%).<br />
The genera with greatest number of species are as<br />
follows: Avenula, Cirsium, Galium, Prunus and<br />
Sambucus – with 2 species each of them (4.08%).<br />
With the highest percentage of coverage (5) are<br />
Quercus cerris L. and Poa pratensis L. (3). With<br />
coverage 2b are Avenula compressa (Heuff.) Sauer et<br />
Chmelit., Avenula pubescens (Huds.) Dumort. and<br />
Lolium perenne L. With coverage 2a are Robinia<br />
pseudoacacia L. and Hor<strong>de</strong>um hystrix Roth. With the<br />
lowest percentage of coverage (+) are Crataegus<br />
monogyna Jacq., Cirsium arvense (L.) Scop. and<br />
Euphorbia agraria Bieb. Only with one individual (r)<br />
is Celtis australis L. Each of the remaining 38 species<br />
has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 34 species (69.39%). Secondly, are annual<br />
herbaceous plants (a) with 10 species (20.41%).<br />
Thirdly, are the shrubs (sh) with 5 species (10.20%).<br />
The trees (t) are 4 species (8.16%). The transition<br />
group of shrubs to trees (sh-t) has 3 species (6.12%).<br />
The biennial herbaceous plants (b) are 2 species<br />
(4.08%). The transition group of annual to biennial<br />
herbaceous plants (а-b) has 1 species (2.04%) only.<br />
The largest number of species (20) has<br />
circumboreal origin. Secondly, are species with<br />
European (11) and Mediterranean origin (10). The<br />
cosmopolitan are 3 species. Two of the species are<br />
adventive elements. One of the species has Pontic<br />
origin. One of the species is Balkan suben<strong>de</strong>mite –<br />
Galium pseudoaristatum Schur.<br />
One species is Tertiary relict in all of the habitat<br />
– Quercus cerris L. It is woo<strong>de</strong>d artificial for the<br />
creation of the shelter belt.<br />
There are no species of protection status. This<br />
can be explained easily by the artificial origin of the<br />
habitat.<br />
39<br />
The anthropophytes are 38 species (77.55%).<br />
The high number is due to the artificial origin of the<br />
habitat and adjacent to farmland.<br />
The anthropogenic influence due to the presence<br />
of: 1. Improved access to the habitat by a system<br />
roads. 2. Grazing goats and cows. 3. Pollution by<br />
garbage from the shepherds and farm workers. 4.<br />
Arable land in the vicinity.<br />
L.<br />
HABITAT 7<br />
Forest shelter belt formed by Fraxinus excelsior<br />
It is located near Chernookovo village,<br />
municipality General Toshevo. The average altitu<strong>de</strong><br />
is 160 m. The exposure is east. The shelter belt is<br />
oriented in a southwest – northeast direction. The<br />
slope is low and reaches 5°. The area is 31.5 dka. The<br />
length of the shelter belt is 2 100 m, the width – 15<br />
m, and the height – 15 m. The soil type is<br />
Chernozems, and the soil subtype is haplic<br />
Chernozems. The bedrock is limestone. The cover of<br />
the tree vegetation is 80%, the cover of the shrub<br />
vegetation is 10% and the cover of the herbaceous<br />
vegetation is 60%.<br />
In the shelter belt have been in<strong>de</strong>tified 59 species<br />
of vascular plants from 50 genera and 19 families.<br />
The families with greatest number of genera are as<br />
follows: Asteraceae – 11 (22.00%), Рosaceae – 6<br />
(12.00%) and Rosaceae – 6 (12.00%). The families<br />
with greatest number of species are as follows:<br />
Asteraceae – 14 (23.73%), Рosaceae – 8 (13.56%)<br />
and Rosaceae – 6 (10.17%). The genera with greatest<br />
number of species are as follows: Chenopodium and<br />
Fraxinus – with 3 species each of them (5.08%);<br />
Artemisia, Bromus, Carduus, Centaurea, Consolida<br />
and Hor<strong>de</strong>um – with 2 species each of them (3.39%).<br />
With the highest percentage of coverage (5) is<br />
Fraxinus excelsior L., followed by Poa pratensis L.<br />
(2а). With the lowest percentage of coverage (+) are<br />
Amorpha fruticosa L. and Elaeagnus angustifolia L.<br />
Only with one individual (r) is Salvia argentea L.<br />
With the cover 1 are 4 species. Each of the remaining<br />
50 species has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 22 species (37.29%). Secondly, are annual<br />
herbaceous plants (a) with 16 species (27.12%).<br />
Thirdly, are the trees (t) with 7 species (11.86%). The
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
shrubs (sh), the biennial herbaceous plants (b) and the<br />
transition group of annual to biennial herbaceous<br />
plants (а-b) have 4 species each of them (6.78%). The<br />
transition group of shrubs to trees (sh-t) has 2 species<br />
(3.39%).<br />
The largest number of species (23) has<br />
circumboreal origin. Secondly, are species with<br />
European (13) and Mediterranean origin (8). The<br />
cosmopolitan are 6 species. Five of the species have<br />
Pontic origin. Four of the species are adventive<br />
elements.<br />
Four species in the habitat are Tertiary relicts:<br />
Acer tataricum L., Cotinus coggygria Scop.,<br />
Fraxinus excelsior L., Quercus cerris L. The main<br />
species is Fraxinus excelsior L. It is woo<strong>de</strong>d artificial<br />
for the creation of the shelter belt. Acer tataricum L.<br />
and Cotinus coggygria Scop. have less than 5%<br />
coverage and their number is more than 50<br />
individuals. The number of the individuals from<br />
Quercus cerris L. is less than 50. Perhaps individuals<br />
of these three species have evolved from fruit, carried<br />
over from adjacent areas.<br />
One species with protection statute is established<br />
– Fraxinus pallisiae Wilmott. It is inclu<strong>de</strong>d in IUCN<br />
Red List for Bulgaria in the category „Vulnerable”. It<br />
has less than 5% coverage, and its number is more<br />
than 50 individuals. The most likely reason for its<br />
presence in the shelter belt is its planting together<br />
with basic species Fraxinus excelsior L.<br />
The anthropophytes are 51 species (86.44%).<br />
Extremely high number of them due to the artificial<br />
origin of the habitat and adjacent to farmland.<br />
The anthropogenic influence due to the presence<br />
of: 1. Improved access to the habitat by a system<br />
roads. 2. Pollution by garbage from the shepherds and<br />
farm workers. 3. Arable land in the vicinity.<br />
HABITAT 8<br />
Forest shelter belt formed by Fraxinus oxycarpa<br />
Willd.<br />
It is located as a third shelter belt between<br />
Vladimirovo and Benkovski villages, municipality<br />
Dobrich. The average altitu<strong>de</strong> is 230 m. The exposure<br />
is northeast. The shelter belt is oriented in a<br />
southwest – northeast direction. The slope is low and<br />
reaches 5°. The area is 17 dka. The length of the<br />
shelter belt is 1 150 m, the width – 15 m, and the<br />
height – 15 m. The soil type is Vertisols, and the soil<br />
40<br />
subtype is eutric Vertisols. The bedrock is limestone.<br />
The cover of the tree vegetation is 80%, the cover of<br />
the shrub vegetation is 10% and the cover of the<br />
herbaceous vegetation is 60%.<br />
In the shelter belt have been in<strong>de</strong>tified 55 species<br />
of vascular plants from 47 genera and 21 families.<br />
The families with greatest number of genera are as<br />
follows: Asteraceae – 9 (19.15%), Rosaceae – 8<br />
(17.02%) and Рosaceae – 6 (12.77%). The families<br />
with greatest number of species are as follows:<br />
Asteraceae – 10 (18.18%), Rosaceae – 10 (18.18%)<br />
and Рosaceae – 7 (12.73%). The genera with greatest<br />
number of species are as follows: Acer and Prunus –<br />
with 3 species each of them (5.45%).<br />
With the highest percentage of coverage (4) is<br />
Fraxinus oxycarpa Willd. With the lowest percentage<br />
of coverage (1) are Amorpha fruticosa L., Tilia<br />
cordata Mill. and Tilia tomentosa Moench. Each of<br />
the remaining 51 species has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 22 species (40.00%). Secondly, are annual<br />
herbaceous plants (a) with 11 species (20.00%).<br />
Thirdly, are the trees (t) with 9 species (16.36%). The<br />
shrubs (sh) and the transition group of shrubs to trees<br />
(sh-t) have 4 species each of them (7.27%). The next<br />
is the transition group of annual to biennial<br />
herbaceous plants (a-b) with 2 species (3.64%). The<br />
biennial herbaceous plants (b), the transition groups<br />
of annual to perennial herbaceous plants (а-р) and of<br />
biennual to perennial herbaceous plants (b-р) are<br />
presented with one species (1.82%) only.<br />
The largest number of species (25) has circumboreal<br />
origin. Secondly, are species with European<br />
(13) and Mediterranean origin (7). The cosmopolitan<br />
are 4 species. Three of the species have Pontic origin.<br />
Three of the species are adventive elements.<br />
Three species in the habitat are Tertiary relicts:<br />
Acer campestre L., Acer tataricum L., Quercus cerris<br />
L. Each of them has less than 5% coverage and their<br />
number is more than 50 individuals. The reason for<br />
their presence can be traced in the transference of<br />
fruit from neighboring areas.<br />
There are no species of protection status. This<br />
can be explained easily by the artificial origin of the<br />
habitat.
Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
The anthropophytes are 45 species (81.82%).<br />
The high number is due to the artificial origin of the<br />
habitat and adjacent to farmland.<br />
The anthropogenic influence due to the presence<br />
of: 1. Improved access to the habitat by a system<br />
roads. 2. Pollution by garbage. 3. Arable land in the<br />
vicinity.<br />
HABITAT 9<br />
Forest shelter belt formed by Gleditsia<br />
triacanthos L.<br />
It is located south of Karapelit village,<br />
municipality Dobrich. The average altitu<strong>de</strong> is 175 m.<br />
The exposure is northeast. The shelter belt is oriented<br />
in a north – south direction. The slope is low and<br />
reaches 10°. The area is 15 dka. The length of the<br />
shelter belt is 1 000 m, the width – 15 m, and the<br />
height – 15 m. The soil type is Chernozems, and the<br />
soil subtype is haplic Chernozems. The bedrock is<br />
limestone. The cover of the tree vegetation is 60%,<br />
the cover of the shrub vegetation is 10% and the<br />
cover of the herbaceous vegetation is 70%.<br />
In the shelter belt have been in<strong>de</strong>tified 88 species<br />
of vascular plants from 78 genera and 30 families. It<br />
is the richest of plant species from the shelter belts.<br />
The reason for this is that is located immediately<br />
adjacent steppe. From the steppe to the shelter belt<br />
migrated a large number of species. The families with<br />
greatest number of genera are as follows: Lamiaceae<br />
– 11 (14.10%), Fabaceae – 9 (11.54%), Rosaceae – 8<br />
(10.26%), Asteraceae – 7 (8.97%), Poaceae – 6<br />
(7.69%) and Apiaceae – 5 (6.41%). The families with<br />
greatest number of species are as follows: Lamiaceae<br />
– 13 (14.77%), Rosaceae – 11 (12.50%), Asteraceae<br />
– 9 (10.23%), Fabaceae – 9 (10.23%), Poaceae – 6<br />
(6.82%) and Apiaceae – 5 (5.68%). The genus with<br />
greatest number of species is Prunus – with 4 species<br />
(4.55%).<br />
With the highest percentage of coverage (4) is<br />
Gleditsia triacanthos L. Secondly, it is Poa pratensis<br />
L. (2а). With the cover 1 are 23 species. With the<br />
lowest percentage of coverage are Carduus<br />
acanthoi<strong>de</strong>s L., Sanguisorba minor Scop. (+) and<br />
Verbascum ovalifolium Sms. (r). Each of the<br />
remaining 60 species has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 42 species (47.73%). Secondly, are annual<br />
herbaceous plants (a) with 16 species (18.18%).<br />
41<br />
Thirdly, are the trees (t) and the shrubs (sh) with 9<br />
species (10.23%) each of them. The next are the<br />
transition group of shrubs to trees (sh-t) and the<br />
biennial herbaceous plants (b) with 4 species each of<br />
them (4.55%). The transition group of annual to<br />
perennial herbaceous plants (а-р) has 2 species<br />
(2.27%). The transition groups of annual to biennial<br />
herbaceous plants (а-b) and of biennual to perennial<br />
herbaceous plants (b-р) are presented with one<br />
species (1.14%).<br />
The largest number of species (28) has<br />
circumboreal origin. Secondly, are species with<br />
Mediterranean origin (21). The next are species with<br />
European (14) and Pontic origin (12). Six of the<br />
species is adventive element. The cosmopolitan are 5<br />
species. One of the species has Oriental-Turanian<br />
origin. One of the species is Balkan en<strong>de</strong>mite –<br />
Achillea clypeolata Sibth. et Sm.<br />
Three species are Tertiary relicts: Celtis australis<br />
L., Cotinus coggygria Scop., Ulmus minor Mill. Each<br />
of them has less than 5% coverage. The number of<br />
Celtis australis L. is less than 50 individuals. The<br />
number of another two species is more than 50<br />
individuals. The reason for their presence can be<br />
traced in the transference of fruit from neighboring<br />
areas.<br />
Four species with protection statute are<br />
established: Adonis vernalis L. is inclu<strong>de</strong>d in CITES<br />
and in the Or<strong>de</strong>r for special arrangements for the<br />
conservation and use of medicinal plants in the<br />
category “Collecting herbs is prohibited from the<br />
natural habitats”. Jurinea le<strong>de</strong>bourii Bunge is<br />
inclu<strong>de</strong>d in the IUCN Red List for Bulgaria in the<br />
category “Endangered”, in the Red book for Bulgaria<br />
in the category „rare” and in the Biological Diversity<br />
Act in the category „protected”. Tilia rubra DC. is<br />
inclu<strong>de</strong>d in IUCN Red List for Bulgaria in the<br />
category „Least Concern” and in the Red book for<br />
Bulgaria in category “Rare”. Asparagus officinalis L.<br />
is inclu<strong>de</strong>d in the the Biological Diversity Act in the<br />
category „Un<strong>de</strong>r the protection and regulated use of<br />
nature”.<br />
The presence of Adonis vernalis L. and Jurinea<br />
le<strong>de</strong>bourii Bunge is associated with their migration<br />
from the nearby steppe region.
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
The anthropophytes are 65 species (73.86%).<br />
The high number is due to the artificial origin of the<br />
habitat and adjacent to farmland.<br />
The anthropogenic influence due to the presence<br />
of: 1. Improved access to the habitat by a system<br />
roads. 2. Pollution by garbage from the shepherds and<br />
farm workers. 3. Arable land in the vicinity.<br />
HABITAT 10<br />
Forest shelter belt formed by Robinia<br />
pseudoacacia L.<br />
It is located as a first shelter belt east of the<br />
highway between Durankulak village and Durankulak<br />
Checkpoint, municipality Shabla. The average<br />
altitu<strong>de</strong> is 20 m. The first half of the shelter belt is<br />
oriented in a west – east direction. The second half of<br />
the shelter belt is oriented in a northwest – southeast<br />
direction. The territory has no inclination. The area is<br />
15 dka. The length of the shelter belt is 1 000 m, the<br />
width – 15 m, and the height – 7 m. The soil type is<br />
Leptosols, and the soil subtype is rendzic Leptosols.<br />
The bedrock is limestone. The cover of<br />
the tree vegetation is 60%, the cover of the shrub<br />
vegetation is 10% and the cover of the herbaceous<br />
vegetation is 80%.<br />
In the shelter belt have been in<strong>de</strong>tified 48 species<br />
of vascular plants from 40 genera and 19 families. It<br />
is the most poor of plant species from the shelter<br />
belts. The families with greatest number of genera are<br />
as follows: Asteraceae – 9 (22.50%) and Рosaceae –<br />
5 (12.50%). The families with greatest number of<br />
species are as follows: Asteraceae – 10 (20.83%),<br />
Рosaceae – 7 (14.58%) and Rosaceae – 6 (12.50%).<br />
The genera with greatest number of species are as<br />
follows: Prunus – 3 (6.25%), Artemisia, Elymus,<br />
Euphorbia, Fraxinus, Galium, Lamium and Poa –<br />
with 2 species each of them (4.17%).<br />
With the highest percentage of coverage (4) is<br />
Robinia pseudoacacia L., followed by species<br />
Elymus repens (L.) Gould. and Elymus hispidus<br />
(Opiz) Meld. (3) and Poa pratensis L. (2а). With the<br />
lowest percentage of coverage (1) are 16 species.<br />
Each of the remaining 28 species has coverage 2m.<br />
The perennial herbaceous plants (p) are most –<br />
they are 19 species (39.58%). Secondly, are annual<br />
herbaceous plants (a) with 9 species (18.75%).<br />
Thirdly, are the trees (t) with 6 species (12.50%). The<br />
next are the shrubs (sh) and the transition group of<br />
42<br />
shrubs to trees (sh-t) with 4 species each of them<br />
(8.33%). The transition group of annual to biennial<br />
herbaceous plants (а-b) has 3 species (6.25%). The<br />
biennial herbaceous plants (b) are 2 species (4.17%).<br />
The transition group of biennual to perennial<br />
herbaceous plants (b-р) has one species (2.08%) only.<br />
The largest number of species (19) has<br />
circumboreal origin. Secondly, are species with<br />
Mediterranean origin (10). The next are species with<br />
european origin – they are 7 species. The<br />
cosmopolitan are 4 species. Three species have<br />
Pontic origin. Three of the species are adventive<br />
elements. One of the species has Oriental-Turanian<br />
origin. One of the species is Balkan suben<strong>de</strong>mite –<br />
Carduus candicans Waldst. et Kit.<br />
In the habitat is meeting once Tertiary relict –<br />
Fraxinus excelsior L. Its coverage is less than 5%.<br />
The number of individuals is in the range 6 – 50. The<br />
reason for its presence can be traced in the<br />
transference of fruit from neighboring areas.<br />
One species with protection statute is established<br />
– Artemisia pe<strong>de</strong>montana Balb. It is inclu<strong>de</strong>d in<br />
IUCN Red List for Bulgaria in the category<br />
„Endangered”, in the Red book for Bulgaria in the<br />
category „Threatened with extinction” and in the<br />
Biological Diversity Act in the category „Protected”.<br />
Its presence can be explained by the transfer of the<br />
fruits by wind and finding favorable conditions,<br />
associated with good light in the shelter belt.<br />
The anthropophytes are 42 species (87.50%).<br />
Extremely high number of them due to the artificial<br />
origin of the habitat and adjacent to farmland.<br />
The anthropogenic influence due to the presence<br />
of: 1. Improved access to the habitat by a system<br />
roads. 2. Arable land in the vicinity.<br />
4. Conclusions<br />
From natural habitats are established most<br />
taxonomical diversity in Western Pontic Paeonian<br />
steppes near to Bejanovo village, and least<br />
taxonomical diversity in Rupicolous calcareous or<br />
basophilic grasslands of the Alysso-Sedion albi<br />
between Onogur and Efreitor Bakalovo villages.<br />
From forest shelter belts are established most<br />
taxonomical diversity in Forest shelter belt formed by<br />
Gleditsia triacanthos L., and least taxonomical<br />
diversity in Forest shelter belt formed by Robinia
Dimcho Zahariev / Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
pseudoacacia L. between Durankulak village and<br />
Durankulak Checkpoint.<br />
The families with greatest number of genera and<br />
species are as follows: Asteraceae, Рoaceae,<br />
Rosaceae and Lamiaceae.<br />
In the analysis of the biological types was<br />
established pattern common to all habitats: most<br />
numerous are perennial and annual herbaceous plants.<br />
This confirms the results of research of Kozuharov et<br />
all. [33].<br />
In all habitats are most floristic elements with<br />
circumboreal, European and Mediterranean origin.<br />
The Tertiary relicts, which are established, are<br />
trees and one shrub. They often have a secondary<br />
origin for the habitats.<br />
The species with protection statute in natural<br />
habitats are most in Western Pontic Paeonian steppes<br />
near to Bejanovo village – they are 8 species. In the<br />
other habitats that number varies from 2 to 4 species.<br />
In the forest shelter belts can be found a small<br />
number of species with protection statute. Most often<br />
they have gone from adjacent areas.<br />
The number of anthropophytes in the natural<br />
habitats is a significant – from 60.13% to 77.67%.<br />
The reasons about this are mainly the following: the<br />
strong fragmentation of natural habitats, arable land<br />
in the vicinity – like source of anthropophytes,<br />
improved access to the habitats and their accessibility<br />
for people and domestic animals. In the forest shelter<br />
belts the anthropophytes quite naturally are more –<br />
from 73.86% to 87.50%.<br />
5. References<br />
[1] VELEV, S., 2002. Climatic zoning, in Kopr<strong>ale</strong>v,<br />
I. (main ed.). Geography of Bulgaria. Physical<br />
and socio-economic geography, Institute of<br />
Geography, BAS, Farkom, Sofia, 760 pp.<br />
[2] NINOV, N., 2002. Soils, in Kopr<strong>ale</strong>v, I. (main<br />
ed.). Geography of Bulgaria. Physical and socioeconomic<br />
geography, Institute of Geography,<br />
BAS, Farkom, Sofia, 760 pp.<br />
[3] KITANOV, B., Penev, I., 1980. Flora of<br />
Dobrudja, Nauka i Izkustvo, Sofia, 630 pp.<br />
[4] BONDEV, I., 1991. The vegetation of Bulgaria.<br />
Map in М 1:600 000 with explanatory text,<br />
University Press St. Kliment Ohridski, Sofia, 183<br />
pp.<br />
43<br />
[5] NATURA 2000 Standard Data Form for<br />
Protected Area „The Valley of Batova River”<br />
(BG0000102), Ministry of Environment and<br />
Waters of Bulgaria, 15 pp.<br />
[6] NATURA 2000 Standard Data Form for<br />
Protected Area „Kraimorska Dobrudja”<br />
(BG0000130), Ministry of Environment and<br />
Waters of Bulgaria, 19 pp.<br />
[7] NATURA 2000 Standard Data Form for<br />
Protected Area „Durankulak Lake”<br />
(BG0000154), Ministry of Environment and<br />
Waters of Bulgaria, 13 pp.<br />
[8] NATURA 2000 Standard Data Form for<br />
Protected Area „Shabla – Ezeretz Lake”<br />
(BG0000156), Ministry of Environment and<br />
Waters of Bulgaria, 16 pp.<br />
[9] NATURA 2000 Standard Data Form for<br />
Protected Area „Suha reka” (BG0002048),<br />
Ministry of Environment and Waters of Bulgaria,<br />
13 pp.<br />
[10] NATURA 2000 Standard Data Form for<br />
Protected Area „Kardam” (BG0000569), Ministry<br />
of Environment and Waters of Bulgaria, 10.<br />
[11] NATURA 2000 Standard Data Form for<br />
Protected Area „Izvorovo – Kraishte”<br />
(BG0000570), Ministry of Environment and Waters<br />
of Bulgaria, 10 pp.<br />
[12] NATURA 2000 Standard Data Form for<br />
Protected Area „Rositza – Loznitza”<br />
(BG0000572), Ministry of Environment and<br />
Waters of Bulgaria, 12 pp.<br />
[13] NATURA 2000 Standard Data Form for<br />
Protected Area „Complex „Kaliakra”<br />
(BG0000573), Ministry of Environment and<br />
Waters of Bulgaria, 27 pp.<br />
[14] TZONEV, R., Rusakova, V., Dimitrov, М.<br />
Dimova, D., Belev, T. Kavrakova, V., 2004.<br />
Proposals for habitats for inclusion to Annex I on<br />
Council Directive 92/43/EEC of the European<br />
Community to protect natural habitats and of<br />
wild fauna and flora and Interpretation handbook<br />
of habitats in the European Union EUR 15/2,<br />
Report, World Wildlife Fund, Danube –<br />
Carpathian Program (WWF, DCP).<br />
[15] KAVRAKOVA, V., Dimova, D., Dimitrov, М.,<br />
Tzonev, R., Belev, T. (editors), 2005. A<br />
guidance for i<strong>de</strong>ntifying the habitats of European<br />
importance in Bulgaria, Geosoft, Sofia, 128.
A characteristic of mo<strong>de</strong>l habitatas in South Dobrudja /Ovidius University Annals, Biology-Ecology Series 14: 33-44 (2010)<br />
[16] KOZUHAROV, S. (ed.), 1992. I<strong>de</strong>ntifier of the<br />
vascular plants in Bulgatia, Nauka i izkustvo,<br />
Sofia, 788 pp.<br />
[17] Flora of PR Bulgaria, Vol. І-Х, 1963-1995,<br />
Publishing House of BAS, Sofia.<br />
[18] CHASE, M. (corresponding author), 2003. An<br />
update of the Angiosperm Phylogeny Group<br />
classification for the or<strong>de</strong>rs and families of<br />
flowering plants: APG II, The Linnean Society of<br />
London, Botanical Journal of the Linnean<br />
Society, 141: 399–436.<br />
[19] PETROVA, А., Anchev, М. Palamarev, Е.,<br />
1999. How to recognize the plants in our nature.<br />
Char i<strong>de</strong>ntifier. Prosveta, Sofia, 837 pp.<br />
[20] WESTHOFF, V., Maarel, E., 1973. The Braun-<br />
Blanquet Approach in: Tuxen, R. (Ed.),<br />
Handbook of vegetation science. Dr. W. Junk b.<br />
v. Publishers the Hague, p. 619-704.<br />
[21] ASIOV B., Petrova A., Dimitrov D., Vasilev R.,<br />
2006. Conspectus of the Bulgarian vascular flora.<br />
Distribution maps and floristic elements,<br />
Bulgarian Biodiversity Foundation, Sofia, 452 p.<br />
[22] GRUEV, B., Kuzmanov B., 1994. General<br />
biogeography, University Press St. Kliment<br />
Ohridski, Sofia, 498 pp.<br />
[23] PEEV, D., 2001. National park Rila.<br />
Management plan 2001 – 2010. Adopted by<br />
Resolution №522 of Council of Ministers on<br />
04.07.2001, Sofia, 338 pp.<br />
[24] BOŽA, P., Anačkov G., Igić R., Vukov D., Polić<br />
D., 2005. Flora “Rimskog šanca” (Vojvodina,<br />
Srbija), 8th Symposium on the flora of<br />
Southeastern Serbia and Neighbouring Regions,<br />
Niš, 20-24.06.2005, Abstracts, рр. 55.<br />
[25] PEEV, D., Kozuharov S., Anchev M., Petrova<br />
A., Ivanova D., Tzoneva S., 1998. Biodiversity<br />
of Vascular Plants in Bulgaria, In: Curt Meine<br />
(ed.), Bulgaria's Biological Diversity:<br />
Conservation Status and Needs Assessment,<br />
Volumes I and II, Washington, D.C.,<br />
Biodiversity Support Program, pp. 55–88.<br />
[26] Council Directive 92/43/EEC of the European<br />
Community to protect natural habitats and of<br />
wild fauna and flora.<br />
[27] Biological Diversity Act, State Gazette number<br />
77 from 9 august 2002, pp. 9–42. Amen<strong>de</strong>d in<br />
State Gazette number 94 from 16.11.2007.<br />
44<br />
[28] Convention on International Tra<strong>de</strong> in<br />
Endangered Species of Wild Fauna and Flora,<br />
State Gazette number 6 from 21 Januari 1992.<br />
[29] Red book of PR Bulgaria, Vol. 1, Plants, 1984,<br />
Publishing House of BAS, Sofia, 447 pp.<br />
[30] PETROVA А., Vladimirov V. (eds.), 2009. Red<br />
List of Bulgarian vascular plants, Phytologia<br />
Balcanica 15 (1): 63–94.<br />
[31] Or<strong>de</strong>r number RD-72 from 3 februari 2006 for<br />
special arrangements for the conservation and<br />
use of medicinal plants, State Gazette number 16<br />
from 21 Februari 2006.<br />
[32] STEFANOV, B., Kitanov B., 1962. Kultigenen<br />
plants and kultigenen vegetation in Bulgaria,<br />
Publishing House of BAS, Sofia, 275 pp.<br />
[33] KOZUHAROV, S., Dimitrov, D., Lazarova, М.,<br />
Kozuharova, Е., 1997. A characteristic of the<br />
flora and the vegetation of the natural plant<br />
complexes in Southern Dobrudja, Conference<br />
proceedings „Dobrudja and Kaliakra”, Aca<strong>de</strong>mic<br />
publishing of Higher Agricultural Institute,<br />
Plovdiv, p. 42-58.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
FLORISTIC ASPECTS OF THE HILLS OF CAMENA VILLAGE<br />
(TULCEA COUNTY)<br />
Marius FĂGĂRAŞ<br />
Ovidius University of Constanţa, Faculty of Natural and Agricultural Sciences, Department of Biology and<br />
Ecology, Mamaia Blvd, No. 124, 900527, Constanţa, Romania, fagarasm@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: This paper presents the flora on the hills in the vicinity of Camena locality. These hills have volcanic<br />
origin and are located in the south-east of the Babadag plateau. The hilly landscape with spectacular rock<br />
formations, the substrate ma<strong>de</strong> up of acidic volcanites and the climate specific to the forest steppe are the main<br />
factors that <strong>de</strong>termined the variety of the vegetation ma<strong>de</strong> up of steppe meadows, rock formations, forests and<br />
bushes. The area is characterized by the presence of a consi<strong>de</strong>rable number of floral rarities, some en<strong>de</strong>mic,<br />
other rare, vulnerable or endangered at national level. Despite all these, the flora of the area is still little known<br />
as there are no specialized papers. The enumeration of the vascular flora is accompanied by an analysis of the<br />
biological forms, of the floral elements, of the ecological categories, but also of the floral rarities present on<br />
these hills.<br />
Keywords: Camena hills, flora, life forms, floristic elements, ecological categories, rare and threatened flora.<br />
___________________________________________________________________________<br />
1. Introduction<br />
The hills of Camena are located south of<br />
Ciucurovei Hills, in the south-east of the Babadag<br />
Plateau, in the vicinity of Camena village (Tulcea<br />
County). They are volcanic hills (Fig. 7), with a<br />
maximum altitu<strong>de</strong> of approx. 190 meters, located at<br />
the southern end of the Peceneaga-Camena crevice<br />
which separates the Northern Dobrogea Plateau<br />
from the Central Dobrogea Plateau. The Hills of<br />
Camena look like a wi<strong>de</strong> saddle framed towards the<br />
north-west and south-east by the hydrographic<br />
basins of two valleys: Camena valley and Ciamurlia<br />
valley. In the southern part of these hills is the<br />
Altan Tepe copper pyrite mine.<br />
The geological layer is ma<strong>de</strong> up of rhyolites<br />
(quartz porphyry) of P<strong>ale</strong>ozoic age, volcanic rocks<br />
(acid volcanites) colored in pink-red, reddish-brown<br />
and violet. In the plane zone and on the ero<strong>de</strong>d<br />
inclines, the rhyolites emerge on the surface as<br />
spectacular rock formations. Towards the base of<br />
the hills, the rocks are covered by a layer of loess<br />
(3-4 meters thick). The soils are represented by<br />
chernozem and lithosoils, the latter being present<br />
especially in the rocky zones.<br />
The climate is temperate-continental, with<br />
average annual temperatures of 10.5-11 0 C, while<br />
the average annual precipitations range between<br />
450 and 500 mm/year. As vegetation type, the Hills<br />
of Camena fit within the forest steppe zone. The<br />
vegetation is ma<strong>de</strong> up of steppe meadows, rock<br />
vegetation (on the plateaus), thermophile forests<br />
and bushes.<br />
The Hills of Camena represent an area of the<br />
Babadag Plateau which is interesting from the<br />
geological, landscape and botanical point of view,<br />
firstly because of the volcanic origin of the hills, of<br />
the rhyolites disposed as spectacular rock<br />
formations and of the floral rarities which can be<br />
encountered in this area. Despite these, the flora of<br />
these hills is little known and limited to the<br />
quotation of species in ol<strong>de</strong>r specialized literature<br />
[1, 2, 3, 4, 5].<br />
2. Material and Methods<br />
The field researches have been done between<br />
years 2008-2010, during the entire vegetation<br />
season in or<strong>de</strong>r to cover all the phenology stages.<br />
The plant taxa nomenclature follows the Flora<br />
ilustrată a României. Pteridophyta et<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
Spermatophyta [6], Flora Europaea [7, 8] and<br />
Flora României [4]. The life forms, floristic<br />
elements and ecological categories have been<br />
established on the base of the synthesis works<br />
Conspectul florei cormofitelor spontane din<br />
România [9] and Flora ilustrată a României.<br />
Pteridophyta et Spermatophyta [6]. The rare and<br />
threatened plant species was done according to the<br />
Romanian Red List [10] and the Romanian Red<br />
Book of the vascular plants [11].<br />
3. Results and Discussions<br />
The floristic researches carried out on the hills<br />
of Camena village have lead to i<strong>de</strong>ntification of 178<br />
vascular taxa (168 species and 10 subspecies)<br />
(Table 1). Taxa found in the studied area belong to<br />
46 families and 38 classes of Spermatophyta<br />
Divisio. The following families are well<br />
represented as number of taxa (Fig. 1): Asteraceae<br />
(12,35%), Lamiaceae (10,67%), Poaceae (9,55%),<br />
Rosaceae si Brassicaceae (5,05%), Liliaceae,<br />
Caryophyllaceae si Fabaceae (cate 4,49%),<br />
Apiaceae (3,93%), Boraginaceae (3,37%),<br />
Ranunculaceae (2,80), Geraniaceae (2,24) and<br />
Scrophulariaceae (1,68%).<br />
14<br />
12<br />
10<br />
8<br />
%<br />
6<br />
4<br />
2<br />
0<br />
AST LAM POA ROS BRAS LIL CARY<br />
families<br />
FAB API BOR RAN GER SCR<br />
Fig. 1. Most important botanical families as the<br />
number of species (AST-Asteraceae; POA-<br />
Poaceae; LAM-Lamiaceae, ROS-Rosaceae; LIL-<br />
Liliaceae;CARY-Caryophyllaceae; FAB-Fabaceae;<br />
API-Apiaceae; BRAS-Brassicaceae;<br />
RAN-Ranunculaceae; BOR-Boraginaceae;<br />
GER-Geraniaceae; SCR-Scrophulariaceae)<br />
From the point of view of the biological forms<br />
(Fig.2), the dominant ones are the<br />
46<br />
hemicryptophytes (42.13%) and the annual and<br />
biennial terophytes (35.95%), present especially in<br />
the steppe meadows. Poorly represented are the<br />
phanerophytes (9.55%), which are inclu<strong>de</strong>d in<br />
forests (with Quercus petraea subsp. d<strong>ale</strong>champii,<br />
Quercus pubescens, Carpinus orientalis, Fraxinus<br />
ornus, Prunus mah<strong>ale</strong>b, Tilia tomentosa) and<br />
bushes (with Crataegus monogyna, Prunus spinosa,<br />
Cotinus coggygria, Ligustrum vulgarae, Rosa<br />
canina, Cornus mas) in the investigated area.<br />
The category of phanerophytes also inclu<strong>de</strong>s<br />
alien species encountered in these hills, some of<br />
them invasive or potentially invasive (Robinia<br />
pseudacacia, Ailanthus altissima, Elaeagnus<br />
angustifolia). The geophytes (7.30%) and the<br />
camephytes (5.05%) are perennial species found<br />
especially in the grassy blanket from forests or<br />
forest edges.<br />
PH<br />
9,55%<br />
TH<br />
35,95%<br />
G<br />
7,30%<br />
CH<br />
5,05%<br />
H<br />
42,13%<br />
Fig. 2. The spectrum of biological forms<br />
(H-hemicriptofite; TH-therofite; PH-fanerofite;<br />
G-geofite; CH-camefite)<br />
Among the floristic elements (Table 2 and<br />
Figure 3), the dominant species are the Eurasian<br />
(35.39%) and Pontic (26.40%) ones, followed at<br />
great distance by other categories of geoelements:<br />
European (8.98%), Central-European (6.17%),<br />
Mediterranean and sub-Mediterranean (6.17%),<br />
Balkan (5.61%), circumpolar (1.68%), Atlantic-<br />
Mediterranean, Taurean-Balkan, Carpatho-Balkan-<br />
Caucasian, and en<strong>de</strong>mic (each with 0.56%).<br />
The large proportion of Pontic species reflects<br />
on the one si<strong>de</strong> the dominance of the steppe<br />
meadows in the studied area, and on the other si<strong>de</strong>,<br />
the nearness of the Razelm-Sinoe lagoon complex<br />
(located approx. 10 km east), which belongs to the<br />
Pontic biogeographic region. Among the categories
Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
of Pontic elements (Fig. 4), the best represented in<br />
the studied area are: the Pontic-Mediterranean<br />
(55.31%), Pontic-Balkan (17.02%), Pontic proper<br />
(10.63%), Pontic-Pannonian-Balkan (8.51%),<br />
Pontic-Pannonian (4.25%), Pontic-Caucasian and<br />
Pontic-Central-European (2.12% each). The arid<br />
climate in the area of these hills is favorable for the<br />
large number of species of southern origin<br />
(Mediterranean, Sub-Mediterranean, Balkan), that<br />
make up a percentage of 11.78%.<br />
Table 2. The percentages of floristic elements<br />
in the studied area<br />
Floristic<br />
elements<br />
Sub-<br />
categories<br />
No. %<br />
Eua 25<br />
Eua Eua(Cont) 17 35,39<br />
Eua(Med) 21<br />
Eur 7<br />
Eur Eur(Cont) 3 8,98<br />
Eur(Med) 4<br />
SE Eur 2<br />
Euc 4<br />
Euc Euc -Med 5 6,17<br />
Euc- 1<br />
subMed<br />
Euc-Balc 1<br />
Pont 5<br />
Pont-Med 26<br />
Pont-Balc 8<br />
Pont Pont-Pan 2 26,40<br />
Pont-Pan- 4<br />
Balc<br />
Pont-Cauc 1<br />
Pont-Euc 1<br />
Med Med 10 6,17<br />
+ subMed subMed 1<br />
Balc 4<br />
Balc-Pan 2<br />
Balc Balc-Anat 1<br />
Balc-Cauc 2 5,61<br />
Balc-Pont- 1<br />
Anat<br />
47<br />
Taur-<br />
Balc<br />
- 1 0,56<br />
Carp-<br />
Balc-<br />
Cauc<br />
- 1 0,56<br />
Atl-Med - 1 0,56<br />
Circ - 3 1,68<br />
End - 1 0,56<br />
Balc<br />
5,61%<br />
Med<br />
6,17%<br />
Pont<br />
26,40%<br />
Cosm<br />
5,05%<br />
Adv<br />
2,24%<br />
Circ<br />
1,68%<br />
Others<br />
2,37%<br />
Euc<br />
6,17%<br />
Eur<br />
8,98%<br />
Eua<br />
35,39%<br />
Fig. 3. The spectrum of floristic elements<br />
Pont-Pan<br />
4,25%<br />
Pont-Pan-Balc<br />
8,51%<br />
Pont-Med<br />
55,31%<br />
Pont-Cauc<br />
2,12%<br />
Pont-Euc<br />
2,12%<br />
Pont<br />
10,63%<br />
Pont-Balc<br />
17,02%<br />
Fig. 4. The spectrum of the Pontic elements<br />
Among the ecological categories connected to<br />
soil humidity (Fig. 5), we can remark the<br />
consi<strong>de</strong>rable percentage of xero-mesophile (57.3%)<br />
and xerophile (24.71%) species, components of the<br />
steppe meadows and of rock formation vegetation.<br />
The mesophile species (14.6%) are present<br />
especially in the forested area of the hills. The<br />
eurythermal species have a smaller percentage<br />
(2.8%).
Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
From the point of view of the preference for<br />
temperature (Fig. 5), the micro-mesothermal<br />
(46.62%) and mo<strong>de</strong>rately thermophile (37.64%)<br />
species have consi<strong>de</strong>rable percentages, as they are<br />
regular in the silvosteppe zone which is ma<strong>de</strong> up of<br />
steppe meadows and forests. The thermophile<br />
species (6.17%) are encountered either in the steppe<br />
meadows or in the rock formations.<br />
From the point of view of the preference for<br />
soil pH (Fig. 5), the higher percentages are held by<br />
poor acid-neutrophile (53.37%), acidic-neutrophile<br />
(17.97%) and euryionic species (20.22%). We must<br />
remark the high percentage of acidophile species<br />
(2.8%), grouped on the acidic volcanites that make<br />
up the rock formations in the plateau zone.<br />
70<br />
60<br />
50<br />
40<br />
%<br />
30<br />
20<br />
10<br />
0<br />
U%<br />
T%<br />
R%<br />
1-1,5 2-2,5 3-3,5 4-4,5 5-5,5 6 0<br />
ecological categories<br />
Fig. 5. The spectrum of ecological categories<br />
The 19 rare and endangered taxa (Table 3)<br />
represent 10.67% of the total species and<br />
subspecies i<strong>de</strong>ntified in the Hills of Camena. A<br />
more important element is the presence of the<br />
en<strong>de</strong>mic species Campanula romanica in the area,<br />
but also of other rare and very rare plants at<br />
national level, mentioned in the Red Book of<br />
vascular plants of Romania [11]: Dianthus<br />
nardiformis (Fig. 8), Silene compacta, Moehringia<br />
jankaea, Iris sintenisii, Salvia aethiopis,<br />
Sempervivum zeleborii, Galanthus plicatus,<br />
Nectaroscordium siculum subsp. bulgaricum,<br />
Achillea coarctata, Crocus reticulatus, etc.<br />
In terms of the main endangered categories<br />
(Fig. 6), 1 taxon (0.56%) is endangered, 4 taxa<br />
(2.24%) are vulnerable, while 14 other (7.86%) are<br />
rare, with small populations at national level.<br />
48<br />
Table 3. The rare and threatened taxa in the<br />
Camena Hills area<br />
No Name of the taxa Floris<br />
tic<br />
eleme<br />
nts<br />
1. Achillea coarctata Pont-<br />
Balc<br />
2. Allium flavum Taursubsp.<br />
tauricum Balc<br />
3. Campanula<br />
romanica<br />
4. Crocus reticulatus Pont-<br />
Med<br />
5. Dianthus<br />
nardiformis<br />
6. Echinops ritro<br />
subsp. ruthenicus<br />
IUCN<br />
categories<br />
[11]<br />
R -<br />
R -<br />
IUCN<br />
categories<br />
[10]<br />
End V/R EN<br />
V -<br />
Balc V/R VU<br />
Pont-<br />
Pan-<br />
Balc<br />
R -<br />
Balc R VU<br />
7. Galanthus<br />
plicatus<br />
8. Iris sintenisii Pont-<br />
Balc<br />
R LR<br />
9 Myrrhoi<strong>de</strong>s<br />
nodosa<br />
Med R -<br />
10 Moehringia Pont R VU<br />
. jankae<br />
11 Nectaroscordium Pont- R -<br />
siculum<br />
bulgaricum<br />
subsp. Balc<br />
12 Paeonia<br />
peregrina<br />
Balc V/R -<br />
13 Salvia aethiopis Pont-<br />
Med<br />
E/R -<br />
14 Sempervivum SE R -<br />
zeleborii<br />
Eur<br />
15 Seseli campestre Pont R -<br />
16 Silene compacta Pont-<br />
Med<br />
R EN<br />
17 Stipa ucrainica Pont-<br />
Cauc<br />
R VU<br />
18 Syrenia cana Pont R -<br />
19 Thymus zygioi<strong>de</strong>s Balc R -
NT<br />
89,33%<br />
Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
E<br />
0,56%<br />
V<br />
2,24%<br />
R<br />
7,86%<br />
Fig. 6. The spectrum of sozological categories<br />
4. Conclusions<br />
The research realized between 2008 and 2010<br />
led to the i<strong>de</strong>ntification of 178 vascular taxa which,<br />
from the taxonomical point of view, belong to 46<br />
families and 38 or<strong>de</strong>rs.<br />
From the point of view of the biological forms,<br />
the dominant are the hemicryptophytes and<br />
terophytes, components of the steppe meadows in<br />
the area of Camenei Hills. The phanerophytes,<br />
camephytes and geophytes are present especially in<br />
the forested areas of these hills. Alongsi<strong>de</strong> the<br />
Eurasian species, well represented in the studied<br />
area are also the Pontic elements specific to west-<br />
Pontic steppes, but also those of southern origin<br />
(Mediterranean, sub-Mediterranean and Balkan), an<br />
expression of a climate with sub-Mediterranean<br />
nuances.<br />
Among the ecological categories of plants<br />
established according to their preference for<br />
substrate humidity, air temperature and soil pH, the<br />
predominant species are xero-mesophile, micromesothermal<br />
and mo<strong>de</strong>rately-thermophile ones, as<br />
well as the poorly acid-neutrophile ones.<br />
Of the total i<strong>de</strong>ntified taxa, the rare and<br />
endangered species represent 10.67%. The<br />
important local populations of certain en<strong>de</strong>mic and<br />
rare species at national level place the Camena Hills<br />
in the northern Dobrogea zones important from the<br />
conservation point of view.<br />
49<br />
5. References<br />
[1] PRODAN I., 1934-Conspectul florei Dobrogei<br />
I, Bul. Acad. <strong>de</strong> Înalte St. Agronomice,<br />
Tipogr. Naţională S.A., Cluj, 5, 1.<br />
[2] PRODAN I., 1935-1936 - Conspectul florei<br />
Dobrogei II, Bul. Acad. <strong>de</strong> Înalte St.<br />
Agronomice, Tipogr. Naţională S.A., Cluj, 6.<br />
[3] PRODAN I., 1938 - Conspectul florei Dobrogei<br />
III, Bul.Facult. <strong>de</strong> Agronomie, Cluj, Tipogr.<br />
Cartea Românească., 7.<br />
[4] SĂVULESCU T. (ed.), 1952-1976 - Flora<br />
României, vol. I-XIII, Edit.Aca<strong>de</strong>miei<br />
Române, Bucureşti.<br />
[5] DIHORU GH., DONIŢĂ N., 1970 - Flora <strong>şi</strong><br />
vegetaţia podişului Babadag, Edit. Aca<strong>de</strong>miei<br />
R.S.R., Bucureşti.<br />
[6] CIOCÂRLAN V., 2000 - Flora ilustrată a<br />
României (Pteridophyta et Spermatophyta),<br />
Edit. Ceres, Bucureşti.<br />
[7] TUTIN T.G. HEYWOOD V.H., BURGES<br />
N.A., MOORE D.M., VALENTINE D.H.,<br />
WALTERS S.M. & WEBB D.A. (eds), 1964-<br />
1980 - Flora Europaea, Vols. 1-5, Cambridge,<br />
Cambridge University Press.<br />
[8] TUTIN T.G. HEYWOOD V.H., BURGES<br />
N.A., MOORE D.M., VALENTINE D.H.,<br />
WALTERS S.M. & WEBB D.A. (eds., assist.<br />
by AKEROYD J.R & NEWTON M.E.;<br />
appendices ed. by MILL R.R.), 1993 (reprinted<br />
1996) - Flora Europaea, 2 nd ed., Vol. 1,<br />
Cambridge, Cambridge University Press.<br />
[9] POPESCU A., SANDA V., 1998 - Conspectul<br />
florei cormofitelor spontane din România, Acta<br />
Botanica Horti Bucurestiensis, Edit.<br />
Universităţii din Bucureşti.<br />
[10] OLTEAN M., NEGREAN G., POPESCU A.,<br />
ROMAN N., DIHORU GH., SANDA V.,<br />
MIHĂILESCU S., 1994 - Lista ro<strong>şi</strong>e a<br />
plantelor superioare din România, Studii,<br />
Sinteze, Documente <strong>de</strong> Ecologie, Bucureşti,<br />
(1): 1-52.<br />
[11] DIHORU GH., NEGREAN G., 2009 - Cartea<br />
Ro<strong>şi</strong>e a plantelor vasculare din România, Edit.<br />
Aca<strong>de</strong>miei Române, Bucureşti.
Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
Fig. 7. General aspect of volcanic hills of Camena<br />
Fig. 8. Dianthus nardiformis on the volcanic rocks<br />
of Camena<br />
50
Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
Table 1. The list of the vascular plants of Camena Hills<br />
No. Taxa<br />
Family Life Floristic Ecological<br />
forms elements categories<br />
1. Achillea coarctata AST H Pont-Balc U1,5 T4,5 R4,5<br />
2. Achillea setacea AST H Eua(cont) U2 T3 R5<br />
3. Adonis flammaea RAN TH Pont-Med U2 T3,5 R3,5<br />
4. Agropyron cristatum subsp. POA H Pont-Euc U2 T4 R4,5<br />
pectinatum<br />
5. Agropyron ponticum POA H(G) Pont-Balc U1,5 T4,5 R4,5<br />
6. Ailanthus altissima SIM PH Adv U0 T0 R0<br />
7. Ajuga chamaepytis subsp. ciliata LAM TH Pont-Med U2,5 T4 R3<br />
8. Ajuga genevensis LAM H Eua(cont) U2 T3 R4<br />
9. Alliaria petiolata LIL TH-H Eua U3 T3 R4<br />
10. Allium flavum subsp. tauricum LIL G Taur-Balc U1,5 T4 R4<br />
11. Allium rotundum LIL G Euc(Med) U2 T4 R4<br />
12 Alyssum alyssoi<strong>de</strong>s BRAS TH Eua(Cont) U1 T3 R0<br />
13. Anagalis arvensis PRIM TH Cosm U3 T3,5 R0<br />
14. Androsace maxima PRIM TH Eua(Cont) U2 T4 R4<br />
15. Anemone sylvestris RAN H Eua(cont) U2 T3,5 R4<br />
16. Anthemis ruthenica AST TH Eur(Cont) U2 T4 R4<br />
17. Anthriscus cerefolium subsp. API TH Pont-Med U2,5 T4 R4<br />
trichosperma<br />
18. Artemisia absinthium AST H(CH) Eua U2 T3 R4<br />
19. Artemisia austriaca AST CH Eua(cont) U2 T4 R4,5<br />
20. Asparagus verticillatus LIL G Pont-Balc U1 T4,5 R4<br />
21. Asperula cynanchica RUB H Pont-Med U2 T3 R5<br />
22. Asperula tenella RUB H Pont-Balc U2 T4 R4<br />
23. Ballota nigra LAM H Euc U2 T3,5 R4<br />
24. Bassia prostrata CHEN CH Eua(cont) U1,5 T4 R4,5<br />
25. Berteroa incana BRAS TH Eua(cont) U2 T3 R4<br />
26. Brachypodium sylvaticum POA H Eua(Med) U3 T3 R4<br />
27. Bromus hor<strong>de</strong>aceus POA TH Eua U0 T3 R0<br />
28. Bromus sterilis POA TH Eua(Med) U2 T4 R4<br />
29. Bromus tectorum POA TH Eua(cont) U1,5 T3,5 R0<br />
30. Buglossoi<strong>de</strong>s arvensis BOR TH Eua U0 T0 R4<br />
31. Buglossoi<strong>de</strong>s purpurocaerulea BOR H-G Euc-subMed U2,5 T4 R4,5<br />
32. C<strong>ale</strong>pina irregularis BRAS TH Pont-Med U2 T4 R3<br />
33. Camelina microcarpa BRAS TH Eua U3 T3 R0<br />
34. Campanula romanica CAMP H End U1,5 T4 R0<br />
35. Campanula sibirica CAMP H Eua(cont) U2,5 T4 R4<br />
36. Cardaria draba BRAS H Eua(Med) U2 T4 R4<br />
37. Carduus acanthoi<strong>de</strong>s AST TH Eur(Med) U2 T3 R0<br />
38. Carpinus orientalis CORY PH Balc-Cauc U3 T4 R4,5<br />
39. Carthamus lanatus AST TH Pont-Med U2,5 T4 R0<br />
40. Centaurea cyanus AST TH Med(Cosm) U3 T4 R0<br />
41. Centaurea diffusa AST TH Pont-Balc U2 T4 R0<br />
51
Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
42. Cerastium brachypetalum CARY TH Med U3 T3 R0<br />
43. Chamomilla recutita AST TH Eua(Med) U2,5 T3,5 R5<br />
44. Chondrilla juncea AST H Eua U1,5 T3,5 R4<br />
45. Chrysopogon gryllus POA H Med U1,5 T4 R4<br />
46. Cichorium intybus AST H Eua U3 T0 R3<br />
47. Conium maculatum API TH-TH Eua U3 T3 R3<br />
48. Convolvulus arvensis CONV H(G) Cosm U2,5 T3,5 R3,5<br />
49. Convolvulus cantabricus CONV H Pont-Med U1,5 T3,5 R4<br />
50. Conyza cana<strong>de</strong>nsis AST TH Adv(Am.N) U2,5 T0 R0<br />
51. Cornus mas CORN PH Pont-Med U2 T3,5 R4<br />
52 Coronilla varia FAB H Eua(Med) U2 T3 R4<br />
53. Corydalis cava FUM G Euc U3 T3 R0<br />
54. Cotinus coggygria ANAC PH Pont-Med U2 T4,5 R4<br />
55. Crataegus monogyna ROS PH Eur U2,5 T3,5 R3<br />
56. Crepis sancta AST TH Pont-Balc U1,5 T4 R4<br />
57. Crocus reticulatus IRID G Pont-Med U2,5 T4 R3<br />
58. Crupina vulgaris AST TH Pont-Med U2 T3,5 R0<br />
59. Cynanchum acutum ASCL H Pont-Med U2,5 T4 R0<br />
60. Cynodon dactylon POA G(H) Cosm U2 T3,5 R0<br />
61. Daucus carota API TH Eua(Med) U2,5 T3 R0<br />
62. Dianthus nardiformis CARY CH Balc U1,5 T4,5 R4,5<br />
63. Dichanthium ischaemum POA H Eua(Med) U1,5 T5 R3<br />
64. Echinops ritro subsp. ruthenicus AST H Pont-Pan-Balc U1,5 T4 R4,5<br />
65. Elaeagnus angustifolia ELEG PH Adv<br />
66. Elymus repens POA G Circ U0 T0 R0<br />
67. Erodium cicutarium GER TH Cosm U2,5 T0 R0<br />
68. Eryngium campestre API H Pont-Med U1 T5 R4<br />
69. Erysimum diffusum BRAS H Eua(Cont) U1,5 T3 R4<br />
70. Euphorbia agraria EUPH H Pont-Med U2 T4 R0<br />
71. Euphorbia nicaeensis EUPH H Pont-Pan-Balc-<br />
Anat<br />
U1,5 T5 R5<br />
72. Festuca v<strong>ale</strong>siaca POA H Eua(cont) U1 T5 R4<br />
73. Filipendula vulgaris ROS H Eua U2,5 T3 R4,5<br />
74. Fragaria viridis ROS H Eur(Cont) U2 T4 R3<br />
75. Fraxinus ornus OLE PH Med U1,5 T3,5 R5<br />
76. Fumaria rostellata FUM TH Euc-Balc U3 T0 R3,5<br />
77. Galanthus plicatus AMAR G Taur-Cauc U3 T4 R3<br />
78. Galium humifusum RUB H Pont-Balc U2 T4 R4,5<br />
79. Geranium divaricatum GER TH Eua(Med) U2,5 T3 R4<br />
80. Geranium pussilum GER TH Eur(Med) U2,5 T3 R0<br />
81. Geranium rotundifolium GER TH subMed U2 T3,5 R4<br />
82. Geum urbanum ROS H Eua(Med) U3 T3 R4<br />
83. Glechoma hirsuta LAM H(CH) Pont-Med-Euc U2,5 T3 R4<br />
84. Hieracium bauhinii AST H Euc U1,5 T3 R3,5<br />
85. Hieracium pilosella AST H Eua U2 T0 R2<br />
86. Holosteum umbellatum CARY TH Eua(Med) U2 T3,5 R0<br />
87. Hypericum perforatum HYP H Eua U3 T3 R0<br />
88. Iris sintenisii IRID G Pont-Balc U2 T4 R4<br />
52
Marius Făgăraş / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
89. Koeleria macrantha POA H Circ U2 T4 R5<br />
90. Lamium amplexicaule LAM TH Eua(Med) U2,5 T3,5 R0<br />
91. Lappula barbata BOR TH-TH Pont-Med U2 T3,5 R4<br />
92. Lathyrus tuberosus FAB H(G) Eua(Med) U2 T4 R4<br />
93. Ligustrum vulgare OLE PH Eua(Med) U2,5 T3 R3<br />
94. Linum austriacum LIN H Eua(cont) U1,5 T3,5 R4<br />
95. Malva sylvestris MALV TH(H) Eua(Cosm) U3 T3 R3<br />
96. Marrubium peregrinum LAM H Eua(Med) U2 T4 R0<br />
97. Marrubium vulgarae LAM H(CH) Eua(Med) U1 T4 R4<br />
98. Medicago lupulina FAB TH(H) Eua U2,5 T3 R4<br />
99. Medicago minima FAB TH Eua(Med) U1,5 T4 R4<br />
100. Melica ciliata POA H Eur(Med) U1,5 T4 R4<br />
101. Melilotus alba FAB TH Eua U2,5 T3 R0<br />
102. Minuartia setacea CARY CH Pont U1,5 T0 R4<br />
103. Moehringia jankae CARY H Pont U1 T4 R4,5<br />
104. Myosotis stricta BOR TH Eua(Med) U2 T0 R2,5<br />
105. Myrrhoi<strong>de</strong>s nodosa API TH Med U2,5 T4,5 R4,5<br />
106. Nectaroscordum siculum subsp.<br />
bulgaricum<br />
LIL G Pont-Balc U3,5 T3,5 R3,5<br />
107. Nigella arvensis RAN TH Pont-Med U2 T4 R4<br />
108. Nonea atra BOR TH Balc-Anat U2 T4 R3<br />
109. Onopordum acanthium AST TH Eua(Med) U2,5 T4 R4<br />
110. Onosma visianii BOR H Pont-Pan-Balc U1,5 T4,5 R4,5<br />
111. Origanum vulgare LAM H Med U2 T3 R3<br />
112. Orlaya grandiflora API TH Euc-Med U2 T3,5 R4<br />
113. Ornithogalum refractum LIL G Balc-Pan-Cauc U2 T3,5 R4<br />
114. Paeonia peregrina PAE H(G) Balc U2 T3,5 R5<br />
115. Papaver dubium PAP TH Eur U2 T3,5 R3<br />
116. Papaver rhoeas PAP TH Cosm U3 T3,5 R4<br />
117. Petrorhagia prolifera CARY TH Pont-Med U1,5 T4 R3<br />
118. Phleum phleoi<strong>de</strong>s POA H Eua(cont) U2 T3 R4<br />
119. Phlomis tuberosa LAM H Eua(Cont) U2,5 T3,5 R4<br />
120. Pinus nigra PIN PH Eua U0 T0 R0<br />
121. Plantago lanceolata PLAN H Eua U3 T0 R0<br />
122. Poa angustifolia POA H Eua U2 T3 R0<br />
123. Polycnemum majus CHEN TH Eua U1,5 T4,5 R4<br />
124. Polygonum aviculare POLG TH Cosm U2,5 T0 R3<br />
125. Polygonatum latifolium LIL G Pont-Pan-Balc U3 T3,5 R4<br />
126. Potentilla argentea ROS H Eua U2 T4 R2<br />
127. Potentilla recta s.l. ROS H Eur(Cont) U1,5 T3,5 R4<br />
128. Prunus mah<strong>ale</strong>b ROS PH Med U2 T3 R4,5<br />
129. Prunus spinosa ROS PH Eur(Med) U2 T3 R3<br />
130. Quercus petraea subsp. FAG PH E.Med.-Carp- U2,5 T2,5 R0<br />
d<strong>ale</strong>champii<br />
Balc<br />
131. Quercus pubescens FAG PH Med U1,5 T5 R5<br />
132. Ranunculus oxyspermus RAN H Balc-Cauc U2,5 T3 R3<br />
133. Reseda lutea RES TH(H) Eua(Med) U2 T3 R0<br />
134. Robinia pseudacacia FAB PH Adv(Am.N) U2,5 T4 R0<br />
53
Floristic aspects of the Hills of Camena village / Ovidius University Annals, Biology-Ecology Series 14: 45-54 (2010)<br />
135. Rosa canina ROS PH Eur U2 T3 R3<br />
136. Rumes acetosella subsp. POLG H SE Eur U2 T3 R2<br />
137.<br />
acetoselloi<strong>de</strong>s<br />
Salvia aethiopis LAM H Pont-Med U2 T5 R0<br />
138. Salvia nemorosa LAM H Pont-Med U2 T4 R4<br />
139. Salvia nutans LAM H Pont-Pan U1 T5 R5<br />
140. Sambucus nigra CAPR PH Eur U3 T3 R3<br />
141. Saxifraga tridactylites SAX TH Eua U2 T3,5 R4<br />
142. Scilla bifolia LIL G Euc U3,5 T3 R4<br />
143. Scleranthus annuus CARY TH-TH Eua U2 T3 R2<br />
144. Scleranthus perennis CARY H(CH) Eur U3 T0 R3<br />
145. Sedum maximum CRAS H Eur U2,5 T0 R4<br />
146. Sempervivum zeleborii CRAS CH SE Eur U1,5 T3,5 R4,5<br />
147. Senecio jacobaea AST H Eua U2,5 T3 R3<br />
148. Seseli campestre API H Pont U2,5 T4 R4<br />
149. Si<strong>de</strong>ritis montana LAM TH Eua U2 T4 R4<br />
150. Silene compacta CARY TH Pont-Med U2 T4 R4<br />
151. Sisymbrium orient<strong>ale</strong> BRAS TH Pont-Med U2,5 T4 R3<br />
152. Solidago virgaurea AST H Circ U2,5 T3 R3<br />
153. Sonchus oleraceus AST TH Cosm U3 T0 R0<br />
154. Stachys germanica LAM H Pont-Med U2 T4 R3<br />
155. Stachys recta LAM H Pont-Med-Euc U2 T5 R5<br />
156. Stipa capillata POA H Eua(Cont) U1 T5 R4<br />
157. Stipa ucrainica POA H Pont-Cauc U1 T4 R4<br />
158. Syrenia cana BRA TH Pont U1,5 T4 R4<br />
159. Teucrium chamaedrys LAM CH Euc(Med) U2 T4 R4<br />
160. Teucrium<br />
capitatum<br />
polium subsp. LAM CH Med U1,5 T4 R4,5<br />
161. Thalictrum minus RAN H Eua U2 T4 R4<br />
162. Thlaspi perfoliatum BRAS TH Eua U2,5 T3,5 R4,5<br />
163. Thymus pannonicus LAM CH Pont-Pan U1,5 T3,5 R4<br />
164. Thymus zygioi<strong>de</strong>s LAM CH Balc U1,5 T4 R4,5<br />
165. Tilia tomentosa TIL PH Balc-Pan U2,5 T3,5 R3<br />
166. Tragopogon dubius AST TH Euc(Med) U2,5 T3,5 R0<br />
167. Trifolium arvensae FAB TH Eua(Med) U1,5 T3 R4<br />
168. Trifolium campestre FAB TH Eur U3 T3 R0<br />
169. Trifolium echinatum FAB TH Med U1,5 T4,5 R4<br />
170. Urtica dioica URT H Cosm U3 T3 R4<br />
171. V<strong>ale</strong>rianella lasiocarpa VAL TH Balc-Pont-Anat U1,5 T5 R4<br />
172. Verbascum phlomoi<strong>de</strong>s SCR TH Euc(Med) U2,5 T3,5 R4<br />
173. Veronica<br />
jacquinii<br />
austriaca subsp. SCR H Pont-Med-Euc U2 T4 R4<br />
174. Veronica teucrium SCR H Eua(Med) U1,5 T4 R4,5<br />
175. Vinca herbacea APOC H Pont U2 T5 R4<br />
176. Vincetoxicum hirundinaria ASCL H Eua(Cont) U2 T4 R4<br />
177. Viola kitaibeliana VIO TH Pont-Med U2 T4 R4,5<br />
178. Viola odorata VIO H Atl-Med U2,5 T3,5 R4<br />
54
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
IDENTIFICATION OF SOME ROSE GENITORS WITH RESISTANCE TO THE<br />
PATHOGENS AGENTS ATTACK<br />
*Marioara TRANDAFIRESCU, Corina GAVĂT, Iulian TRANDAFIRESCU and Elena DOROFTEI<br />
*Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology<br />
Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: mtrandafirescu@yahoo.com<br />
________________________________________________________________________________________<br />
Abstract: In the South-Eastern Romania, as in all country the rose culture is higly praised for its ornamental<br />
value both in parks and domestic gar<strong>de</strong>n. In this zone of our country the rose culture is more important because<br />
the Black Seasi<strong>de</strong> provi<strong>de</strong> a better enviroment (82 km). Besi<strong>de</strong> the growing of forign cultivars from Europe<br />
Companies, Romania has done a breeding work to <strong>de</strong>velop autochtonous cultivars (Rusticana, Ambasador,<br />
Bordura <strong>de</strong> nea, Rosagold, Simina, etc.) better adapted to our local conditions. In rose breeding besi<strong>de</strong>s the<br />
ornamental value of these flowers (nice leaves, colours and shapes) the disease resistance has been taken into<br />
acount. Among the specific pathogens very harmful for the rose culture, one can mention: Sphaerotheca pannosa<br />
(Wallr) Lev var rosae Woron (pow<strong>de</strong>ry mil<strong>de</strong>w), Diplocarpon rosae Wolf (black spott), Phragmidium<br />
mucronatum (Pers) Schlecht (rose rust) and Botrytis cinerea Pers (grey mold). One of the most effective methods<br />
to prevent these pathogens attack is breeding new cultivar and genically resistant genitors. These paper present<br />
the behaviour of 50 genotypes from rose collection of Fruit Growing Development of Fruit Tree Constanta and<br />
their response of such pathogens. The conditions of natural infections allowed grouping the biological material in<br />
4 classes of resistance. This clasifications was done acording to levels of frequency (F%) and intensity (I). The<br />
rose cultivars with genetic resistance to this pathogens are: Queen Elisabeth, Foc <strong>de</strong> tabara, Rubin, Parfum,<br />
Emerald d’or, Bel Ange, Apogee.<br />
Keywords: black spott, pow<strong>de</strong>ry mil<strong>de</strong>w, rose rust, genitors, resistance<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
From the ol<strong>de</strong>st times the rose was consi<strong>de</strong>red<br />
"Queen of The Flowers" due its beauty, perfume,<br />
richness in colors and multiple shapes of the grown<br />
cultivars. Therefore, the rose place is in the front of<br />
the ornamental species used for park and gar<strong>de</strong>ns<br />
<strong>de</strong>coration and for cut-flowers production.<br />
Unfortunately, the rose as many other cultivated<br />
plants can suffer due to the attack of some very<br />
damaging pathogens. Un<strong>de</strong>r favorable conditions, the<br />
pathogens can <strong>de</strong>termine the partial or total<br />
<strong>de</strong>foliation of the plants, they become weak and the<br />
cut-flowers production can be diminished by quantity<br />
and quality as well.<br />
In or<strong>de</strong>r to prevent and control the pathogens<br />
specific to the roses, the studies carried out in<br />
Romania and in the World, were focused on<br />
i<strong>de</strong>ntification of the species responsible for the<br />
diseases occurrence and knowledge of their biology<br />
(Bedian, 1980, Bernardis, 2004, Ostaciuc, 1982,<br />
Sandu 2004, Szekelly, 1981, Wagner, 2002), and on<br />
the other hand, was investigated the efficacy of some<br />
fungici<strong>de</strong>s in or<strong>de</strong>r to control them(Bon and coll,<br />
1978, Morrison, 1978, Hagan and coll 1988, Losing,<br />
1988, Qvarnstrom, 1989, Raabe, 1989, Rolim and<br />
col, 1990, etc).<br />
The results obtained in the World (Saun<strong>de</strong>rs,<br />
1970; Klimenko, 1973; Simonyan, 1973; Semina,<br />
1980, 1984; Palmer, 1978; Costlediene, 1981) and in<br />
our country (Costache, 1993, Argatu, 1993, Sekely,<br />
1981, Wagner, 2002) clearly emphasized that the<br />
most efficient method to prevent the attack of the<br />
pathogens is the creation and extension in the culture<br />
of some roses cultivars genetically resistant to<br />
diseases.<br />
Therefore, the researches carried out during<br />
2008-2009 at Research Station for Fruit Growing<br />
ISSN-1453-1267 © 2010 Ovidius University Press
I<strong>de</strong>ntification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010)<br />
Constanta had as central objective the evaluation of<br />
some roses cultivars behavior to some key pathogens<br />
in or<strong>de</strong>r to i<strong>de</strong>ntify some resistance donors genitors<br />
for further breeding works.<br />
In the reference area the pathogens of economic<br />
importance for the rose culture are Diplocarpon<br />
rosae Wolf, Sphaerotheca pannosa (Wallr)Lev var<br />
rosae Woron <strong>şi</strong> Phragmidium mucronatum (Pers)<br />
Schlecht.<br />
2. Material and Methods<br />
The biological material for investigations was<br />
represented by 50 roses cultivars preserved in the<br />
collection owned by Research Station for Fruit<br />
Growing Constanţa. Observation were ma<strong>de</strong><br />
regarding the attack frequency (F%) and intensity (I<br />
notes) of the pathogens Diplocarpon rosa,<br />
Sphaerotheca pannosa var rosae and Phragmidium<br />
mucronatum, and finally the attack <strong>de</strong>gree (AD) was<br />
calculated<br />
For disease intensity (I notes) the sc<strong>ale</strong> „0-6”<br />
was used. The observations were carried out in the<br />
period of attack maximum for each of three key<br />
pathogens studied.<br />
According the attack <strong>de</strong>gree (A.D.) value, the<br />
cultivars were classified in five resistance classes as<br />
follow:<br />
- resistant (R) cu A.D.= 0-5%<br />
- slightly attacked (SA) with A.D. = 5.0-12.5%<br />
- medium resistant (MR) with A.D. = 12.5-22.5%<br />
- sensible (S) with A.D. = 22.5-37.6%<br />
- very sensible (VS) with A.D. = more than 37,6%<br />
To establish the D.L., in or<strong>de</strong>r to establish the<br />
five classes of cultivars the average value took into<br />
calculations ws 22.9%.<br />
3. Results and Discussions<br />
In 2008, the attack of Diplocarpon rosae, fungi<br />
which <strong>de</strong>termine the black spot disease was<br />
evi<strong>de</strong>nced on rose cultivars in the third <strong>de</strong>ca<strong>de</strong> of<br />
May, being stimulated by the amount of precipitation<br />
at the vegetation start (114,9 mm) and by the high<br />
atmospheric relative humidity (over 75%).<br />
The disease symptoms occurrence on leaves<br />
consisted in some black spots, between 2-5 mm up to<br />
56<br />
10-18 mm in diameter, highly visible on the superior<br />
face of the leaves. (Fig.1).<br />
Fig. 1. Pătarea neagră a frunzelor <strong>de</strong> trandafir –<br />
Diplocarpon rosae<br />
Un<strong>de</strong>r such condition, presented in table 1, the<br />
(natural) genetic resistance to the pathogen attack<br />
manifested the cultivars: Emeran<strong>de</strong> d’or, Grad<br />
Premiere, Traviata, Apogee, Luchian, Monica, Coup<br />
<strong>de</strong> Foudre and Tour Eiffel.<br />
The cultivars: Baccara, Pascali, Bel Ange,<br />
Creole, Dame <strong>de</strong> Coeur and Ingrid Bergman vere<br />
evi<strong>de</strong>nced as slightly attacked (SA); and the cultivars:<br />
Flamenco, Karla, Rumba, Maria Callas, Grand Mogol<br />
and Montezuma were evi<strong>de</strong>nced as medium resistant<br />
(MR).<br />
Very sensible (V.S) leaves black spot attack<br />
proved to be the cultivars Mainzer Fastnacht, Rose<br />
Gaujard, Kor<strong>de</strong>s Perfecta, Detroit, Horido <strong>şi</strong> Konigin<br />
<strong>de</strong>r Rosen, but they can be used as indicators for this<br />
disease. At this group of cultivars the plants were<br />
premature <strong>de</strong>foliated at the end of July.<br />
The climatic conditions from the Black Sea<br />
coast, characterized by strong wind, high temperature<br />
during the day (26-28 o C) and the presence of the<br />
water con<strong>de</strong>nse on the vegetative organs of the plants<br />
favorised, the occurrence of the pow<strong>de</strong>ry mil<strong>de</strong>w<br />
attack produced by Sphaerotheca pannosa var rosae<br />
fungi starting with the last <strong>de</strong>ca<strong>de</strong> of May.<br />
The symptoms were noticed initially on the both<br />
si<strong>de</strong>s of the leaves as irregular white dusty spots<br />
(Fig.2).
Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010)<br />
Table 1. Behaviour of some cultivars roses to the<br />
attack of the main pathogens agents<br />
CULTIVARS Diplocarpon Sphaerotheca Phragmidium<br />
rosae pannosa var. rosae mucronatum<br />
G.A. Resistan G.A. Resistan G.A. Resistan<br />
(%) -ce class (%) -ce class (%) -ce class<br />
Broca<strong>de</strong> 7.2 S.A. 11.6 M.R. 12.3 M.R.<br />
Grand premiere 0 R 0 R 4.6 S.A.<br />
Creole 12.4 S.A. 6.5 S.A. 3.2 S.A.<br />
Traviata 0 R 1.2 R 14.6 M.R.<br />
Grand prix 32.4 S 0 R 0 R<br />
Horido 47.3 F.S. 1.0 R 3.6 R<br />
Apogee 0 R 0.8 R 0 R<br />
Luchian 0 R 4.2 S.A. 3.6 S.A.<br />
First love 1.2 R 17.6 M.R. 38.4 F.S.<br />
Concerto 3.7 R 9.7 S.A. 12.2 S.A.<br />
Konigin <strong>de</strong>r<br />
Rosen<br />
63.0 F.S. 14.2 M.R. 0 R<br />
Foc <strong>de</strong> tabără 4.1 R 1.6 R 1 R<br />
Miss Univers 26.3 S 0 R 0 R<br />
Chicago Peace 32.0 S 37.6 F.S. 22.8 S<br />
Kronenburg 34.6 S 17.6 M.R. 0 R<br />
Bel Ange 4.3 S.A. 0 R 0 R<br />
Cocotte 0 R 0 R 18.3 M.R.<br />
Samurai 1.6 R 3.6 R 4.3 R<br />
Monica 0 R 0 R 0 R<br />
Montezuma 17.4 M.R. 9.6 S.A. 4.5 S.A.<br />
Don Juan 3.6 R 0 R 0 R<br />
Grand Mogol 15.0 M.R. 2.0 R 42.6 F.S.<br />
Sutter’s Gold 11.7 S 42.6 F.S. 53.2 F.S.<br />
Effel Tour 0 R 3.6 S.A. 1.2 R<br />
Detroit 37.9 F.S. 6.4 S.A. 0 R<br />
Maria Callas 15.6 M.R. 0 R 1.2 R<br />
Simfonia albă 24.0 S 0 R 1.2 R<br />
Mabella 57.0 F.S. 10.2 M.R. 13.7 M.R.<br />
Pascali 12.2 S.A. 0 R 0.9 R<br />
Rumba 19.3 M.R. 6.9 S.A. 22.0 M.R.<br />
King’s Ranson 0 R 1.2 R 54.6 F.S.<br />
Baccara 6.3 S.A. 22.0 S 7.2 S.A.<br />
Superstar 1.2 R 3.6 R 12.6 M.R.<br />
Kor<strong>de</strong>s Perfecta 51.2 F.S. 17.9 M.R. 21.3 M.R.<br />
Madame<br />
Meilland<br />
0.7 R 7.3 S.A. 4.6 S.A.<br />
Coup Foudre 0 R 12.6 M.R. 17.2 M.R.<br />
Mr. Lincoln 2.6 R 0 R 0 R<br />
Dame <strong>de</strong> Coeur 7.2 S.A. 12.6 M.R. 42.1 F.S.<br />
Rose Gaujard 42.6 F.S. 51.8 F.S. 22.4 M.R.<br />
Carina 12.4 S.A. 6.3 S.A. 1.2 R<br />
Queen Elisabeth 3.2 R 1.2 R 0 R<br />
Eminance 1.2 R 2.6 R 0 R<br />
Mainzer<br />
Fastnacht<br />
38.6 F.S 7.9 S.A. 9.6 S.A.<br />
Karla 13.0 M.R. 1.6 R 2.0 R<br />
Flamenco 19.3 M.R. 3.6 R 12.3 S.A.<br />
Ingrid Bergman 7.2 S.A. 14.6 M.R. 1.2 R<br />
Ambassador 1.2 R 6.2 S.A. 1.6 R<br />
Emeran<strong>de</strong> d’or 0.6 R 38.6 F.S. 0 R<br />
Parfum 0 R 1.8 R 2.6 R<br />
Rubin 0 R 0 R 0 R<br />
57<br />
Fig. 2. Făinarea trandafirului – Sphaerotheca<br />
pannosa var. rosae<br />
Afterwards, the attack progressed covering<br />
almost entirely the leaves, which turn in yellow, then<br />
dried and fallen down. In this case the attack<br />
progressed also on the young floral buds of the<br />
sensible cultivars, which were covered by the<br />
mycelium felt and they could not open.<br />
Assessment of the data presented in the same<br />
table revel that, a high (natural) resistance to this<br />
damaging pathogen stroke manifested the cultivars:<br />
Grand Prix, Miss Univers, Maria Callas, Pascali,<br />
Simfonia albă, their vegetative organs were entirely<br />
clear from Sphaerotheca pannosa var rosae. fungi<br />
symptoms.<br />
At the other pole were the cultivars: Emeran<strong>de</strong><br />
d’or, Chicago Peace, Sutter’s Gold and Rose Gaujard<br />
which were rated as very sensible (V.S.), but they can<br />
be used as sensibility indicators.<br />
The attack produced by Phragmidium<br />
mucronatum fungi, was noticed at the end of the first<br />
<strong>de</strong>ca<strong>de</strong> of May and progressed until the last <strong>de</strong>ca<strong>de</strong> of<br />
September. In this month this pathogen attack<br />
frequency (F%) and the intensity (I notes) registered<br />
the highest values.<br />
From the beginning the disease progressed on<br />
all plants organs: leaves, young branches, stalks and<br />
floral buds (Fig. 3). On these organs was noticed the<br />
presence of some orange pustules representing the<br />
fungus ecidia.
I<strong>de</strong>ntification of some rose genitors with resistance... / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010)<br />
Fig. 3. Rugina trandafirului – Phragmidium<br />
mucronatum<br />
In the last <strong>de</strong>ca<strong>de</strong> of May, on the inferior face of<br />
the plant leaves, small p<strong>ale</strong>-yellow pustules occurs,<br />
representing the nest of uredospores which produce<br />
repeated secondary infections during the vegetation<br />
period.<br />
Starting with the second <strong>de</strong>ca<strong>de</strong> of June, on the<br />
inferior face of the plant leaves, was observed the<br />
presence of the black pustules representing the shelter<br />
of the teleutospores containing the resistance organs<br />
of the fungus.<br />
Among the cultivars that manifested a<br />
pronounced genetic resistance to the attack of this<br />
pathogen can be mentioned: Apogee, Bel Ange,<br />
Emeran<strong>de</strong> d’or, Grand Prix, Kroenenburg, Kroningin<br />
<strong>de</strong>r Roson, Miss Univers, Detroit, Rubin, Queen<br />
Elisabeth, Eminance. Their vegetative organs were<br />
entirely clear from pathogen signs all of the<br />
vegetation period.<br />
The vast majority of the other cultivar studied<br />
showed themselves as slightly attacked (SA) or<br />
medium resistant (MR).<br />
In the case of this pathogen, as was highlighted<br />
in table 1, very sensible cultivars (VS) manifested the<br />
cultivars Grand Mogol, Sutter’s Gold, King’s Ranson<br />
and First love.<br />
4. Conclusions<br />
In the Romanian zone of Black Sea coast, the<br />
pathogens with economical importance for the roses<br />
grown in open fields are: Diplocarpon rosae Wolf,<br />
Sphaerotheca pannosa (Wallr) Lev var rosae Woron<br />
<strong>şi</strong> Phragmidium mucronatum (Pers) Schlecht.<br />
The rose cultivars Emerald d’or, Bel Ange,<br />
Apogee, Foc <strong>de</strong> tabără, Queen Elisabeth, Rubin,<br />
Parfum and Rubin, present genetic resistance for all<br />
three damaging agents and can be used as resistance<br />
genitors in the works carried out to bread new disease<br />
resistant rose cultivars.<br />
The fact that un<strong>de</strong>r the some climatic<br />
conditions, the rose cultivars manifest various attack<br />
<strong>de</strong>grees to the pathogens reveals that, the resistance is<br />
cultivars trait, which represent the key factor in<br />
prevention of the most damaging specific pathogens.<br />
58<br />
5. References<br />
[1] BEDIAN G., 1980. Rust (Phragmidium sp.) on<br />
roses, R.P.P., 59, 4, 1562.<br />
[2] BON Y., Bourdin J., Berthier G., 1978. Efficacité<br />
<strong>de</strong> quelques fongici<strong>de</strong>s vis-á-vis <strong>de</strong> L’oidium du<br />
rosier (Sphaerotheca pannosa var. rosae),<br />
Phytiatrie – Phytopharmacie, 27 (3), 199-205.<br />
[3] CASTLENDINE P., Grout B.W.W., Roberts<br />
A.V., 1981. Cuticular resistance to Diplocarpon<br />
rosae, Transaction of the British Mycological<br />
Society, 47.<br />
[3] COSTACHE C., Costache M., Argatu Constanta,<br />
1993. Rezultate preliminare privind comportarea<br />
unor soiuri <strong>de</strong> trandafir la atacul principalilor<br />
agenţi patogeni. An<strong>ale</strong>le I.C.L.F. vol. XII, 119-<br />
129.<br />
[4] HAGAN A. K., Gillian C. H., Fare D. C.,1987.<br />
Evaluation of new fungici<strong>de</strong>s for control of rose<br />
black spot, Journal of Environmental Horticulture<br />
6 (2), 67-69.<br />
[5] LÖSING H., 1988. Bekämpfung von Rosenrost,<br />
Deutsche Baumschule 40 (11), 518-519.<br />
[6] MORRISON L. S., 1978. Preliminary results on<br />
the evaluation of fungici<strong>de</strong>s for the control of<br />
black spot of rose. Nursery Research Field Day P<br />
– 777, 59-60.<br />
[7] QVARNSTRÖM K., 1989. Control of black spot<br />
(Marssonina rosae) on roses, Växtskyddsnotiser<br />
53 (3), 58-63.<br />
[8] PALMER L. T., Salac S. S., 1978. Reaction of<br />
several types of roses to black spot fungus,<br />
Diplocarpon rosae, Indian Phytopathology 30<br />
(3), 366-368.<br />
[9] ROLIM P. R. R., Toledo A. C. D., Cardoso R.<br />
M. G., Brignani Neto F., Oliveira D. A., 1990.<br />
Comparison of fungici<strong>de</strong>s for control of rose<br />
black spot (Diplocarpon rosae) and pow<strong>de</strong>ry<br />
mil<strong>de</strong>w (Sphaerotheca pannosa var. rosae),<br />
Summa Phytopathologicals 16 (3-4), 269-274.<br />
[10] SAUNDERS P. J. W., 1970. The resistance of<br />
some cultivars and species of Rosa to<br />
Diplocarpon rosae Wolf causing black spot<br />
disease, Natn. Rose, A., 118-128.<br />
[11] SEMINA S. N., Klimenco Z. K., 1976.<br />
Evaluation of gar<strong>de</strong>n rose gene pool for
Marioara Trandafirescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 55-59 (2010)<br />
resistance to pow<strong>de</strong>ry mil<strong>de</strong>w. Byull Gosudar,<br />
Nikit. Bot. Sada, 2 (30), 48-54.<br />
[12] SIMONYAN S. A., 1973. Pow<strong>de</strong>ry mil<strong>de</strong>w of<br />
rose in the Erevan Botanical Gr<strong>de</strong>n. Biol. J.<br />
Armenii, 26 (7), 62-73.<br />
[13] SZEKELY I., Wagner Şt., Drăgan Maria, 1981.<br />
Rezistenţa diferitelor soiuri <strong>de</strong> trandafir faţă <strong>de</strong><br />
atacul <strong>de</strong> făinare (Sphaerotheca pannosa var<br />
rosae) în funcţie <strong>de</strong> unele caracteristici anatomomorfologice,<br />
Simpoz. CAER Cluj, ASAS, ICPP.<br />
[14] WAGNER Şt., Râureanu V., 1996. Princip<strong>ale</strong>le<br />
boli <strong>şi</strong> dăunători ai trandafirilor <strong>şi</strong> combaterea<br />
lor. Rosarium, nr. 1.<br />
59
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
PRELIMINARY DATA ON MELEDIC – MANZALESTI NATURAL RESERVE (BUZAU<br />
COUNTY, ROMANIA)<br />
Daciana SAVA *, Mariana ARCUŞ**, Elena DOROFTEI *<br />
*Ovidius University, Natural Sciences Faculty,<br />
Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania, e-mail: daciana.sava@gmail.com<br />
** Ovidius University, Faculty of Pharmacy, Aleea Universitatii No. 1, corp B, Constanţa, 900470, Romania<br />
__________________________________________________________________________________________<br />
Abstract: the Meledic –Manz<strong>ale</strong>sti Reserve is situated in the central-eastern part of Romania, in Buzau County,<br />
60km north form town of Buzau. The Reserve (136 hectares) is <strong>de</strong>limitated by four rivers and it is situated at a<br />
medium altitu<strong>de</strong> of 530 m. Because of the remarkable forms of relief, appeared as a result of dissolution of salt, the<br />
presence of a salt cave unique in Europe and of a number of lakes with fresh water, this area was <strong>de</strong>clared in 1986<br />
Geological and Speological Reserve. Later (in 2000) due to the presence of an interesting flora and fauna it was<br />
established the value of its natural heritage, and was <strong>de</strong>clared as „Protected Natural Area” with geological,<br />
speological, floral and faunistic importance. In 2007 it was <strong>de</strong>clared „Site of Community Importance” and will<br />
become area of special conservation after the validation of the European Commission. The present study took place<br />
over a period of two years, with field trips in various periods of the year. As for the flora, taxons belonging to over<br />
100 genera were i<strong>de</strong>ntified, Most genera belong to Fabaceae, Asteraceae, Labiatae, Rosaceae and Umbelliferae<br />
families. The statistical analysis showed as biological forms, the predominance of hemicrytophytes. As floristic<br />
elements, the Euro-Asiatic and Central European elements predominated. As regarding the ecological preferences<br />
(humidity, temperature and soil reaction) it has been observed the domination of xeromesophytic, mezothermal and<br />
euriionical species.<br />
Keywords: Natural Reserve, Manz<strong>ale</strong>sti –Meledic Natural Reserve, Romania<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
In 1978, a group of Romanian spelologists, part of<br />
“Emil Racoviţã” Speologists Club from Bucharest,<br />
discover in the sub-Carpathians Mountains at<br />
Mânzãleşti, the longest (300 m), the <strong>de</strong>epest (44 m)<br />
and the most ramificated cavity in salt in the country,<br />
second in the world as oscillation of level, third as<br />
length, wich they named “The cave with three<br />
entrances“ from Sãreni. In 1980 the “6s“ Cave from<br />
Mânzãleşti is discovered, the longest cave in salt<br />
world-wi<strong>de</strong> at that moment (1257 m) and the second as<br />
oscillation of level (-32 m), with numerous<br />
ramifications. Later on, other galleries, were<br />
discovered of a total of 4257 m length, 32 caves<br />
digged in salt, taking the Mânzãleşti cave to the<br />
second place in the world for caves digged in carst of<br />
salt.<br />
Later on, in 1986, the salt carst from<br />
Mânzãleşti becomes a reserve of The Romanian<br />
Aca<strong>de</strong>my from the geological and speleological point<br />
of view.<br />
According to the Habitat Directive 92/43/CEE<br />
concerning the conservation of natural habitats, wild<br />
flora and fauna, the protected areas network Natura<br />
2000 appears in România, which inclu<strong>de</strong>s also in this<br />
network the Meledic Plateau of Mãnzãleşti commune,<br />
Buzãu County, according to Law nr 5/5 March 2000<br />
[1].<br />
The site has the ROSCI 0199 co<strong>de</strong> and is<br />
classified un<strong>de</strong>r category IV (according to UICN) as<br />
Special Conservation Area. The reserve is part of the<br />
Continental Biogeographical Region; its existance is<br />
trying to protect the “Ponto-sarmatic <strong>de</strong>ciduous<br />
thickets” habitats.<br />
Characterization of the “Meledic Plateau”<br />
Reserve<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Preliminary data on Meledic-Manz<strong>ale</strong>sti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010)<br />
„Meledic Plateau” Reserve is situated in the sub-<br />
Carpathians Mountains, in the Lopãtari Dingle and in the<br />
Slãnic river superior basin (tributary streamof Buzãu<br />
river) (latitu<strong>de</strong> N 45 ο 29’ 49” and longitu<strong>de</strong> of E 26 ο 37’<br />
16”). The reserve has a length of 1.7 km, on the North-<br />
South axis and 1.2 km on the West-East direction,<br />
holding a total surface of 157 hectares, the plateau being<br />
situated at an altitu<strong>de</strong> between 400m and 600m.<br />
In the Eastern si<strong>de</strong>, the <strong>de</strong>limitation is <strong>de</strong>termined by<br />
the Jgheab river (a tributary stream of the Slãnic river),<br />
North of the Meledic stream and West of the Sãrat<br />
stream, the latter tributary stream of the Jgheab Valley,<br />
which emptyes into the Sãrat stream in turn. Out of these<br />
rivers and streams only the Meledic stream has fresh<br />
water, the other streams being also alimented with salty<br />
springs, their water having a brackish taste.<br />
Relief<br />
The Meledic Plateau Slopes are very abrupt,<br />
allowing sometimes to see the structure of the plateau<br />
represented by a layer of clay and sh<strong>ale</strong> on the upper<br />
si<strong>de</strong>, with a thickness of 10 to 30 meters, un<strong>de</strong>r which<br />
there is the block of salt, tall up to a few hundred meters.<br />
The Meledic Reserve represents one of the most<br />
unprece<strong>de</strong>nted places, the relief is expanding on the salt<br />
located on the surface or shallow <strong>de</strong>pth, resulting one of<br />
the most interesting regions in our country. A very<br />
diversified terrain in shape and size <strong>de</strong>velops because of<br />
the dissolution of salt on slopes (Fig.1, Fig.2).<br />
Fig.1. Aspect of the abrupt slopes in Plateau<br />
Meledic Reserve (south view)<br />
62<br />
Fig.2. Aspect of the abrupt slopes in Plateau<br />
Meledic Reserve (vest view)<br />
On the western si<strong>de</strong> we can notice blocks of salt<br />
integrated in clay and salty sh<strong>ale</strong>, on which gaps and<br />
limestones have <strong>de</strong>veloped, in comparison with the<br />
southern si<strong>de</strong> where vein of salt can be noticed even on<br />
the surface. Where the salty water rivers come out on<br />
the surface arises a rapid vaporisation of the water<br />
resulting in especially beautiful salt cristals.<br />
The plateau is located on the upper si<strong>de</strong> of the<br />
reserve and is crossed by sinkholes, closed dingles,<br />
oval or round, with a diameter that can reach<br />
sometimes 40 m and a <strong>de</strong>pth of 25 m, wi<strong>de</strong>r dingles<br />
results by their blending.<br />
On the bottom of such sinkholes, where the salt<br />
was covered with a <strong>de</strong>nser layer of clay, freshwater<br />
lakes were formed, receiving water only from rain or<br />
snow meltdown. These lakes have karstic origins, all<br />
the un<strong>de</strong>rground springs are salt watered, the<br />
connection with these ending long time ago. The<br />
presence of freshwater lakes on a salt massif is<br />
consi<strong>de</strong>red a unique phenomenon.<br />
Soils<br />
In the Meledic reserve we encounter a large<br />
variety of soils.<br />
On the steep slopes, where the salt layers are very<br />
close to the surface or even on the surface, we find the<br />
white alkali. On the slopes where the water carries<br />
small amounts of silt the vertisols are formed, on<br />
heavy clay rocks (with high clay content).<br />
On the plateau we meet halomorphous soils,<br />
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)<br />
partially covered with sediment surfaces. A great<br />
diversity of halophile species <strong>de</strong>velop here.<br />
In sinkholes where the sedimented clay have<br />
allowed the formation of lakes, the soils are<br />
hydromorphic, formed due to an excess of moisture,<br />
which can be permanent or temporary. In areas where<br />
the water has a permanent stagnation, the pseudogleic<br />
soil is formed. The plant species <strong>de</strong>veloping here are<br />
thermophilic.<br />
In forested areas we can find reddish brown<br />
forest soils, especially clay soils. In these areas we<br />
especially find Pontic or Mediterranean species.<br />
Climate<br />
The sub-Carpathians Mountains have a<br />
temperate-continental climate, with regional<br />
differences imposed by the shape of relief, but also by<br />
the position at the intersection of climate influences<br />
northwest, eastern and southern.<br />
Being located in a lowland area of the sub-<br />
Carpathians, the Meledic Plateau has a low hill<br />
climate with a ten<strong>de</strong>ncy of aridity in summer. The<br />
average annual temperatures fit between 6 ο C and 8 ο<br />
C. The average annual temperature of the col<strong>de</strong>st<br />
month, January, is of 3 ο C, and of the hottest month,<br />
July, is of 18 ο C.<br />
The average annual rainfall is around 700-800<br />
mm. The largest amount of rainfall is in May and<br />
June, and the driest months are September and<br />
October.<br />
2. Material and Methods<br />
Field trips have been organized for the flora<br />
studies in the Meledic Reserve from Mânzãleşti: two<br />
trips in the months of May and June (months that<br />
have a rapid vegetation <strong>de</strong>velopment), and in the<br />
months of April, July, August, September only one<br />
trip, through the years 2007-2008 to catch the<br />
different stages of vegetation (vernal and estival).<br />
After rating, we ma<strong>de</strong> up a floristic list, in which<br />
the plants have been placed in the right systematic<br />
units [1, 2, 3]. Based on this list, we ma<strong>de</strong> out: the<br />
systematic analysis of the vegetation, the bioform<br />
spectrum, the geoelements spectrum, the spectrum for<br />
ecological preferences: humidity, temperature and<br />
soil reaction [4, 5].<br />
63<br />
3. Results and Discussions<br />
Due to the field trip a total of 133 taxa were registered.<br />
The following taxa were i<strong>de</strong>ntified in the study area: Acer<br />
campestre L. Ph (MM); Eur.; U2,5 T3 R3, Achillea<br />
millefolium L. H; Euras.; U4 T3 R0 , Adonis aestivalis L.<br />
Th; Euras; U3T4 R3, Agrimonia eupatoria L. H; Euras.; U2,5<br />
T3 R4, Ajuga genevensis L. H; Euras.; U2,5 T3 R4 , Alisma<br />
plantago-aquatica L. HH; Cosm.; U6 T0 R0, Alnus incana<br />
(L) Moench Ph (MM); Eur.;U4 T2 R4, Alnus viridis<br />
(Chaix.) DC Ph (MM);Alp.-eur;U3,5 T2,5R3, Anchusa<br />
officinalis L. limba boului); TH; Eur.; U2 T3,5 R0, Anemone<br />
nemorosa L. G; Eur; U3,5 T4 R0, Anemone ranunculoi<strong>de</strong>s<br />
L. G; Eur; U3,5 T3 R4, Artemisia vulgaris L. H; Circ.;U3 T3<br />
R4, Astragalus onobrychis L. H; Euras.; U1,5 T3,5R4,5,<br />
Ballota nigra L. Th; Centr. Eur.); U3 T3,5 R0, Betonica<br />
officinalis L .(Stachys officinbalis L.) H; Euras.; U3 T3 R3,<br />
Brassica rapa L.Th; Med; U3 T3 R4, Campanula<br />
rapunculoi<strong>de</strong>s L. H; Euras.; U3 T2 R0, Capsela bursapastoris<br />
Medicus Th; Cosm; U3 T0 R0, Carex digitata L.<br />
H; Euras.; U3 T3R3, Carum carvi L. TH; Euras.; U3,5 T3 R3,<br />
Centaurea spinulosa Roch. H; Centr. Eur.; U2,5 T0 R3,<br />
Centaurea nervosa Willd. H; Alp.-eur.;U3 T0 R3;<br />
Centaurium umbellatum Gilib. Th; Centr.eur.;U3 T3 R2,<br />
Chaerophyllum bulbosum L. TH; Centr. Eur; U4 T3,5 R4,5,<br />
Chrysanthemum leucanthemum L. H; Euras.; U 3T3,5 R3,<br />
Chrysanthemum corymbosum L. H; Euras.; U3 T3 R3,<br />
Clematis vitalba L. Ph ; Centr. Eur.; U3 T3 R3, Colchicum<br />
autumn<strong>ale</strong> L. G; Eur; U3,5 T3 R4, Coronilla varia L. H;<br />
Centr. Eur.; U2 T3 R4, Cornus mas L. Ph (M); Pont. medit.;<br />
U2 T3,5 R4, Cornus sanguinea L. Ph (M);Centr. Eur); U3 T3<br />
R4, Corylus avellana L. Ph (M); Eur.; U3 T3 R3, Crataegus<br />
monogyna Jacq. Ph (M); Euras.;U2,5 T3 R3, Cytisus<br />
hirsutus L. Ph (N); Centr. Eur.; U2,5 T3 R2, Daucus carota<br />
L. TH; Euras.; U2,5 T3 R0, Delphinium consolida S.F.Gray<br />
(Consolida regalis) Th; Euras; U3 T4 R4, Diplotaxis muralis<br />
L. Th; Centr. Eur; U2,5 T3,5 R4, Draba verna Chevall Th;<br />
Euras; U2,5 T3,5 R0, Echium vulgare L. TH; Euras.; U2 T3<br />
R4, Elaeagnus angustifolia L. Ph (M); Euras; U0 T3 R4,5,<br />
Epipactis atropurpurea Raf G; Euras.; U2 T0 R4,5,<br />
Equisetum arvense L. G.; Cosm.; U3 T3 R0, Erigeron<br />
cana<strong>de</strong>nsis L. Th; Adv.; U2,5 T0 R0, Eryngium campestre<br />
L. H; Pont. medit.; U1 T5 R4 , Euphorbia cyparissias L. H;<br />
Eur.; U2 T3 R4, Euphrasia rostkoviana Hayne. Th; Centr.<br />
Eur.; U3 T3 R3, Fagus sylvatica L. Ph (MM); Centr. Eur.;<br />
U3 T3 R0, Festuca pratensis Hudson H; Euras; U3,5 T0 R0,<br />
Ficaria verna L. (Ranunculus ficaria Huds.) H;Euras: U3,5
Preliminary data on Meledic-Manz<strong>ale</strong>sti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010)<br />
T3 R3, Filipendula ulmaria Maxim. H; Euras: U4,5 T2<br />
R0, Fragaria viridis Duch. H; Euras.; U2 T4 R3,<br />
Fraxinus ornus L. Ph (M); Medit.; U1,5 T3,5 R5, Gagea<br />
pratensis Dumort. G; Eur; U2 T3 R3, Galanthus nivalis<br />
L. G; Centr. Eur.; U3,5 T3 R4, Galium verum L. H;<br />
Euras.; U2,5 T2,5 R0, Galium vernum Scop. H; Euras; U3<br />
T2 R2, Hippophae rhamnoi<strong>de</strong>s L. Ph (M); Euras.; U0 T3<br />
R4,5, Hypericum perforatum L. H; Euras.; U3 T3 R0,<br />
Juniperus communis L. Ph (M); Circ.; U2 T0 R0,<br />
Knautia arvensis (L.) Coult. H; Eur.; U2,5 T3 R0,<br />
Knautia silvatica Duby. H; Centr. Eur; U2 T3 R0, Larix<br />
<strong>de</strong>cidua Miller Ph (MM); Carp; U2,5T0 R0, Lathyrus<br />
tuberosus L. H; Euras.; U2 T4 R4, Lathyrus pratensis L.<br />
H; Euras; U3,5 T3 R4, Leontodon hispidus L. H;<br />
Euras.;U2,5 T0 R0, Lepidium draba Desv. H; Euras: U2<br />
T4 R4, Linum austriacum L. H; Euras.; U1,5 T3,5 R4,<br />
Lithospermum purpureo-caeruleum L. H; CentrEur;U2<br />
T3,5 R4, Lythrum salicaria L. H; Circ.; U4 T3 R0,<br />
Medicago lupulina L. Th ; Euras.; U2,5 T3 R4, Medicago<br />
falcata L.Th; Euras.; U2 T3 R4, Melampyrum arvense L.<br />
Th; Eur.; U2 T3,5 R4,5 , Melilotus officinalis (L.) Pallas<br />
Th; Euras.; U2,5 T3,5 R0, Morus nigra L. Ph (MM); Adv;<br />
U2 T3,5 R4, Muscari comosum (L. ) Miller G; Eur.; U1,5<br />
T3,5 R0, Myosotis sylvatica Hoffm. H; Euras: U3,5 T3 R3,<br />
Onobrychis viciifolia Scop.H; Euras.; U2 T3 R0 , Orchis<br />
purpurea Huds. G; Centr. Eur.;U 2,5 T4 R4,5, Origanum<br />
vulgare L. H; Euras.;U2,5 T3 R3, Orlaya grandiflora L.<br />
Th; Med; U2 T3,5 R4, Ornithogalum refractum Kit. G;<br />
Balc-Pan- Cauc; U2 T3,5 R4, Phragmites australis<br />
Steu<strong>de</strong>l HH; Cosm.; U5 T0 R4, Picea excelsa Link Ph.<br />
(MM); Centr. Eur.; U0 T0 R0, Pinus sylvestris L.<br />
Ph.(MM); Euras.; U0 T0 R0, Plantago media L. H;<br />
Euras; U2,5 T0 R4,5, Poa pratensis L. H; Circ: U3 T0 R0,<br />
Polygala amara L. H; Eur.; U0 T2 R4,5, Polygala major<br />
Jacq. H; Pont. –medit.; U2 T3 R4,5, Potamogeton natans<br />
L. HH; Cosm.; U6 T 2,5 R4, Potentilla reptans L. H;<br />
Cosm; U3,5 T0 R4, Potentilla argentea L. H; Euras; U2T4<br />
R2, Primulla officinalis Hill. H; Euras.;U3 T2 R5,<br />
Prunella vulgaris L. H ; Circ. U3 T3 R0, Prunella<br />
grandiflora (L.) Scholler H; Eur.; U3 T3 R4,5, Prunus<br />
cerasifera Ehrh. Ph (M); Euras: U2 T4 R0, Pyrus<br />
piraster Burgsd. Ph (M); Eur; U2 T3 R4, Quercus<br />
d<strong>ale</strong>champii Ten. Ph (MM); Medit; U2,5 T3 R0,<br />
Ranunculus arvensis L. Th; Euras.; U3 T3 R0,<br />
Rhinanthus minor L. Th; Eur.; U3 T0 Ro, Rosa canina<br />
L. Ph (N); Eur.; U2 T3 R3, Rubus caesius L. Ph (N);<br />
Eur.; U2 T3 R4, Salix alba L. Ph (MM); Euras; U5 T3 R4,<br />
Salix caprea L. Ph (M); Euras; U3 T3 R3, Salix<br />
64<br />
pentandra L. Ph (MM); Euras.;U4,5 T0 R3,5, Salvia<br />
verticillata L. H; Medit.;U2 T4,5 R4, Salvia nemorosa L. H;<br />
Centr. Eur.; U2,5 T4 R3, Scabiosa ochroleuca L. H; Euras.;<br />
U2 T4 R4, Schoenoplectus tabernaemontani (Gmelin)<br />
Palla HH;Euras.; U5,5 T4 R5, Senecio vernalis Waldst et.<br />
Kit.<br />
Th; Euras.;U2,5 T4 R0, Silene vulgaris Garke H; Euras: U3<br />
T3 R4, Sinapis arvensis L. Th; Euras.; U3 T4 R4,<br />
Sisymbrium sophia Webb. Th; Euras; U2,5 T4 R4, Stachys<br />
lanata Jacq. H; Medit.; U2 T0 R0, Thlaspi perfoliatum L.<br />
Th; Euras; U2,5 T3,5 R4,5, Thymus glabrescens Willd. Ch;<br />
Pont.-pan.; U2 T4 R0, Tilia cordata Miller Ph (MM); Eur.;<br />
U3 T3 R3, Tragopogon pratensis L. H; Euras.; U3 T2 R3,<br />
Trifolium campestre Schreb. Th; Eur; U3 T3 R0, Trifolium<br />
medium L. H; Euras.; U3 T3 R0, Typha angustifolia L.<br />
HH; Circ.; U6 T4 R0, Ulmus laevis Pall. (velniş); Ph (MM);<br />
Eur.; U4 T3 R3, Veronica chamaedrys L. Ch; Euras.; U3 T0<br />
R0, Veronica arvensis L. Th; Eur; U2,5 T3 R,Veronica<br />
teucrium L Ch; Euras.; U1,5 T4 R4,5, Vicia angustifolia L.<br />
Th; Euras.; U2 T3 R 0, Vicia sepium L. H; Euras.; U3 T3 R3,<br />
Vicia cracca L. H; Euras; U3 T0 R3, Vicia hirsuta S.F.<br />
Gray. Th; Euras; U2.5 T3,5 R4, Viola arvensis Murr. Th;<br />
Cosm.; U3 T3 R0, Viola hirta L. H; Euras.; U2 T3 R4, Viola<br />
tricolor L. Th; Euras.; U2,5 T3 R0.<br />
Statistic flora analysis<br />
Taxa found in the reserve belong to 4 classes, 43<br />
families and 133 species. The largest number of species has<br />
the following families: Fabaceae (16 species), Labiatae,<br />
Rosaceae and Compositae (10 species each),<br />
Scrophulariaceae, Umbelliferae (5 species each), the other<br />
families were represented by only 1, 2, or 3 species each.<br />
Analysis of the biological forms<br />
Analyzing the spectrum of the biological forms we<br />
discover that in the reserve hemicryptophyte dominate<br />
(41%) from the species i<strong>de</strong>ntified. These are followed<br />
(21%) by the phanerophytes which together with therophites<br />
(24%) form another 43% from the biological forms, the rest<br />
being represented by the geophytes and the helohydrophytes<br />
(Fig.3).
4%<br />
8%<br />
20%<br />
2%<br />
Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010)<br />
4%<br />
21%<br />
41%<br />
H Ph Ch G HH Th TH<br />
Fig.3. Analysis of the biological forms<br />
Analysis of the floristic elements<br />
The analysis of the floristic elements mark out the<br />
dominant euro Asiatic elements, which among those<br />
central European sum up approximately 82 species<br />
(70%) from the reserve flora, forming more than half the<br />
floral elements which means that they constitute the<br />
floristic background of this reserve.<br />
Mediterranean and Ponto Mediterranean floristic<br />
elements, which are thermophile species found especially<br />
on the sunny slopes, form together 8%. However, the<br />
cosmopolitic species are also remarkable, representing<br />
5% from the total species found in the reserve.<br />
The fact that the reserve in situated in sloppy area is<br />
confirmed by the presence of the circumpolar and even<br />
alpine European at the reserve level, together<br />
representing 7% of the total species (Fig.4).<br />
46%<br />
1%1%1% 5%<br />
5%<br />
2%<br />
1%<br />
5%<br />
2%<br />
14%<br />
14%<br />
Daco.Balc Carp. Balc.Pan. Cauc Circ<br />
Cosm Adv Eur Centr.Eur<br />
Alp Pont Pan Medit Euras<br />
Fig.4. Analysis of the floristic elements<br />
Ecologic study of the cormophytes<br />
Humidity<br />
If we group the plants by their humidity regimen in<br />
which they are adjust to live here, we will discover that<br />
the most dominant are xeromesophilic (U2-U2,5) which<br />
65<br />
are 42% from the total of the i<strong>de</strong>ntified species in the<br />
reserve, these being found in the droughtiest places,<br />
specially on the meadows.<br />
Notable are also the mesophilic (U3-U3,5) which have<br />
a percentage of 37%, and can be found in areas where the<br />
light is scarce or there is an excess in humidity, where the<br />
swamps dry up during the summer but nevertheless have an<br />
excess in moisture.<br />
The xerophilic (U1-U1,5) can be found in 5% and this<br />
shows the hot and arid summer climate, being especially<br />
noticed on the slopes that have a south exposure, covered<br />
with a small seam of clay soil.<br />
In a percentage of 6%, the mesophilical (U4-U4,5)<br />
species that prefer soils from humid to moist-wet, are found<br />
near lakes or where there is an excess in moisture all year<br />
long.<br />
Remarkable is the presence of the hydrophilic (U5-<br />
U5,5) and ultrahydrophilic (U6) species which together<br />
form 5% from the total of species, and can be found in the<br />
ponds or on their bor<strong>de</strong>r where water is present all year<br />
round.<br />
The amphytoletant (U0) species can also be found in<br />
the reserve in a percentage of 6%, being the most adaptable<br />
for these special conditions (Fig.5).<br />
22%<br />
20%<br />
4%<br />
1%<br />
5%<br />
2% 1% 2% 2%<br />
4%<br />
28%<br />
U3 U3,5 U4 U2,5 U5 U5,5 U6 U0 U1 U1,5 U2,5 U2.5<br />
Fig. 5. Ecological spectrum of humidity<br />
Temperature<br />
Mesothermal (T3-T3,5) appear in a percentage of<br />
63%. Mild thermophilic (T4-T4,5) species appear in a<br />
percentage of 12%, which suggests that the climate in the<br />
reserve in a temperate-continental one.The<br />
amphylotolerant (T0) appear in 16% of the total.<br />
The cryophilic (T1) species are missing, and the<br />
microthermal (T5) species appear in a small percentage,<br />
only 1% (Fig.6).<br />
9%
Preliminary data on Meledic-Manz<strong>ale</strong>sti Reserve... / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010)<br />
45%<br />
2%<br />
6%<br />
17%<br />
1% 1%<br />
14%<br />
45%<br />
14%<br />
T3 T3,5 T4 T4,5 T5 T0 T2 T2,5 T3<br />
Fig.6. Ecological spectrum of temperature<br />
Soil reaction<br />
37% of the i<strong>de</strong>ntified species are euroionic (R0);<br />
poor acid-neutrophilic species (R4-R4,5) are<br />
presented in the same proportion; acid-neutriphilic<br />
species (R3-R3,5) can be found in a percentage of 21%,<br />
and the neutrophilic-basophilic (R5) are found only in a<br />
percentage of 3%. The balance of the acidophilic plants<br />
(R2) is just 2%, and those highly acidophilic (R1) are<br />
missing (Fig.7).<br />
34%<br />
2%<br />
10%<br />
3%<br />
20%<br />
R3 R3,5 R4 R4,5 R5 R0 R2<br />
30%<br />
Fig. 7. Ecological spectrum for soil reaction<br />
4. Conclusions<br />
The flora in the reserve is highly diversified, being<br />
represented by the distribution of the species in 43<br />
families, predominant being the families Fabaceae,<br />
Labiatae, Compositae.<br />
Hemicryptophyte (41%) appear in the highest<br />
percentage indicating the presence of the herbal<br />
evergreen species, adaptable to the edapho-climatic<br />
conditions in the areas. Therophites (20% + 4%) are<br />
1%<br />
66<br />
plants mostly found in the northern regions, with an arid<br />
climate, and are presented in the reserve through the<br />
annual or biannual species. Phanerophytes (21%)<br />
presented by trees and scrubs, indicate the presence of<br />
forests in the reserve, as well as the cover of the slopes<br />
with scrubs which assure its stabilization.<br />
The analysis of the floristic elements reveals the<br />
predominant Euro-Asian elements, which along the central<br />
European totalize approximately 82 species (70%) from<br />
the reserve flora, representing the floristic background of<br />
the reserve. Floristic Mediterranean and Ponto<br />
Mediterranean elements, which are theomophilic species,<br />
can be found on<br />
the sunny slopes. The fact that the reserve in situated in a<br />
hill area can also be acknowledged because of the<br />
circumpolar and even alpine European species found here.<br />
The presence of xeromesophitic species (U2-U2,5) in a<br />
percentage that represents almost half of the total of the<br />
i<strong>de</strong>ntified species in the reserve (42%), indicate a arid<br />
climate which is specially caracteristical for the medows.<br />
The mesophilic species (U3-U3,5 ), which have a pretty<br />
high percentage (37%), can be found in the areas where the<br />
light is scarce or it is excessively moisturized or in the<br />
areas where the swamps completely dry out during<br />
summer, but remain excessively moisturized. The<br />
mesothermal (T3-T3,5) along the mild thermophilic (T4-<br />
T4,5) are presented in a higher percentage, which mean that<br />
the climate in the reserve is a temperate continental one.<br />
Regarding the distribution according to the reaction of the<br />
soil, the euroionic species (R0) can be found in a pretty<br />
high percentage (34%), almost equal to those of the<br />
species and the poor acid-neutrophilic (R4- R4,5) (40%).<br />
Remarkable is also the presence of the acid-neutrophilic<br />
species (R3- R3,5) (21%) which have a percentage that is<br />
worth taking into account; the presence in the reserve of<br />
different types of habitats: rivers (with fresh and salt<br />
water), lakes, meadows, forests.<br />
Studies are to be done in the future to analyze the<br />
interesting and diverse flora of the region.<br />
5. References<br />
[1] MOHAN GHE, ARDELEAN A., GEORGESCU M.,<br />
1993 - Rezervaţii <strong>şi</strong> monumente <strong>ale</strong> naturii din<br />
România, Casa <strong>de</strong> Editurã <strong>şi</strong> Comerţ, București, 201<br />
pp.
Daciana Sava et al. / Ovidius University Annals, Biology-Ecology Series 14: 61-66 (2010)<br />
[2] BELDIE AL., 1977- Flora României -<br />
Determinator ilustrat al plantelor vasculare, vol. I-<br />
II, Editura Aca<strong>de</strong>miei R.S.R, 406 pp.<br />
[3] CIOCÂRLAN V., 2000 - Flora ilustratã a<br />
României, Editura Ceres, Bucureşti, 1138 pp.<br />
[4] DONIŢÃ N., IVAN D., 1975 - Meto<strong>de</strong> practice<br />
pentru studiul ecologic <strong>şi</strong> geografic al vegetaţiei:<br />
112-331, Editura Didacticã <strong>şi</strong> Pedagogicã,<br />
Bucureşti.<br />
[5] SANDA V., POPESCU A., DOLTU I., DONIŢÃ<br />
N., 1983 - Caracterizarea ecologicã <strong>şi</strong><br />
fitocenologicã a speciilor spontane din flora<br />
României, “Ecological and phytocoenologyical<br />
characterisation of the spontaneous species in<br />
Romanian flora” in: Nat.Scienc.Suppl. 25,<br />
Stud.Communic. Muz. Brukental, Sibiu, 126 pp.<br />
67
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
CONTRIBUTIONS TO THE BIOMETRICAL AND PHYTOBIOLOGICAL STUDY<br />
ON WILD GARLIC<br />
Mariana LUPOAE*, Dragomir COPREAN*, Rodica DINICĂ**,Paul LUPOAE***<br />
* Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences<br />
Street Mamaia, nr. 124, Constanţa, 900527, România, mariana_lupoaie@yahoo.com<br />
** Dunărea <strong>de</strong> Jos University Galaţi, Faculty of Science, Street Domnească no. 47, 800008, Galaţi,<br />
rodinica@ugal.ro<br />
*** Natural Sciences Museum Complex Galaţi-Botanical Gar<strong>de</strong>n, Street Regiment 11 Siret no. 6A, 800340<br />
Galaţi, paul_lupoae@yahoo.com<br />
_____________________________________________________________________________________<br />
Abstract: The purpose of our study was the biometrical and phytobiological analysis of leafs and bulbs on wild<br />
garlic. This species grows spontaneously in the Romanian flora and was harvested for obtaining the drugs on<br />
Măcin Mountains (Luncavita Forest), at altitu<strong>de</strong>s of 150÷200m. By macroscopic examinations in different<br />
phenophasis established in the area study exten<strong>de</strong>d population with Allium ursinum L. ssp. ucrainicum Kleopow<br />
et Oxner (Fam. Alliaceae). The biometrical calculation have been performed according to the literature, early<br />
spring, in months february, march, april and may of year 2010. Leaves finesse expressed by l/L is different:<br />
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<br />
cm². Average mass bulbs = 2,4 g/buc and length by 12 mm to 50 mm.<br />
Keywords: wild garlic; Allium ursinum L. subsp. ucrainicum; leafes and bulbs; biometry.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The Allium genus inclu<strong>de</strong>s approximately 500<br />
species spread worldwi<strong>de</strong>. Allium ursinum L. is a<br />
monocots on family Alliaceae and is wi<strong>de</strong>ly in<br />
Europa, Asia Minor, Caucasus, Siberia up to the<br />
Kamchatka Peninsula.<br />
In Romania this species ”mezohigrofita” grows<br />
in frequent clusters at the shadows of the trees. It has<br />
elliptical-lanceolat leaves with white flowers grouped<br />
and from the biochemical point consist through the<br />
presence of the ether oils with sulfur, that are giving<br />
their own smell [1-3].<br />
Un<strong>de</strong>r various popular names- buckrams, wild<br />
garlic, broad-leaved garlic, wood garlic, sremuš or<br />
bear's garlic- this species is used by locals in<br />
preparations for spring salad and is very appreciated<br />
for many qualities.<br />
They have been shown to have applications as<br />
antimicrobial, antithrombotic, antitumor,<br />
hypolipidaemic, antiarthritic and hypoglycemic<br />
agents [4-8].<br />
The last researches about the population of A.<br />
ursinum from Romania put in evi<strong>de</strong>nce differences of<br />
biomass <strong>de</strong>pending of the geographic area and the<br />
local pedoclimatic conditions.<br />
Wild garlic is a plant which grows on soils with high<br />
mineral trophicity and takes place into the<br />
“megatroph” category with the value V= 85-100 %<br />
[9].<br />
The opportunity of this biometrical and<br />
phytobiological studies consist in the representation<br />
of some morphologic-bulbus,folium,flores-by wild<br />
garlic elements harvested from the Luncavita Forest<br />
(Macin Mountains), a plant with high pharmaceutic<br />
potential.<br />
2. Material and Methods<br />
The harvesting of the bilological material that<br />
was realized with the agreement of the O.S. Macin<br />
that manages the area of the Luncavita Forest<br />
(U.P.I.”Izvorul lui Gavrila”).<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010)<br />
The macroscopic exam served as a review of the<br />
observed characters with free eye or with the<br />
magnifer and as sensory through the perception of the<br />
smell and the taste on the informations contained in<br />
the bibliography of speciality and own researches<br />
[10, 11].<br />
The biometrical observations were obtained<br />
based on the published biometrical calculation<br />
methods. The surface of the leaves was measured<br />
with the help of a mathematic mo<strong>de</strong>l by the summing<br />
of the geometrical figures distributed uniformly on an<br />
sample of 30 leaves [12].<br />
Some representative examples in<strong>de</strong>ntified on<br />
the area are stored in the Herbarium of Botanical<br />
Gar<strong>de</strong>n Galati and of the Pharmacy and Medicine<br />
Faculty of “Dunarea <strong>de</strong> Jos” University Galati.<br />
3. Results and Discussions<br />
A.ursinum grows on big areas in the Luncavita<br />
Forest only in north hills or near the water.<br />
Sometimes this species can be found in other zones<br />
but in low population.<br />
The acompaining flora is composed by different<br />
species like: Corydalis solida, Asarum europaeum,<br />
Corylus avellana, Tilia tomentosa, He<strong>de</strong>ra helix,<br />
Polygonatum latifolium, Scilla bifolia, Carpinus<br />
betulus, Viola odorata, Ranunculus ficaria, Lamium<br />
purpureum, Galium aparine, Geum urbanum,<br />
Anthriscus cerefolium, Muscari botryoi<strong>de</strong>s and<br />
others.<br />
Our observations show us that un<strong>de</strong>r the shrubs<br />
(Corylus avellana) known for its organic requestsrich<br />
soils,<strong>de</strong>ep,loose- and a rich litter,wild garlic<br />
reaches the base of the shrubs [14].<br />
Also,the power of growing and penetration of<br />
the wild garlic was noticed even through the<br />
woody,half<strong>de</strong>scomposed fragments (Fig. 1).<br />
Simple bulbs or two united can be found at a<br />
relative <strong>de</strong>pth small in the soil (3-5cm) especially in<br />
the humus layer and they have good <strong>de</strong>veloped roots<br />
and branched by 3÷15cm length (Fig.2). The<br />
appearance of the leaves are leveled:first in March,<br />
the second simultaneous with the third (by case). The<br />
most of the plants have two leaves.<br />
68<br />
Fig. 1. The escape of the wild garlic through<br />
the wood fragments ( original photo)<br />
Biometric analysis consists of the following<br />
items (Table 1): the leaf length (L), the leaf width (l),<br />
the petiole length (Lp), the percentage ratio-leaf<br />
finesse (l/L), number ribs (R), mass of green leafs<br />
(M).<br />
On the bulbs (Table 2, Fig. 4) were measured<br />
the length (L), the mass (M), diameter (D) and<br />
number of the roots (No.roots).<br />
Our biometric studies ma<strong>de</strong> on the leaves of<br />
A.ursinum shou that the rapport between the width<br />
and the length of the limb leaf is conversely<br />
proportional with the procedure of growing (Fig.3):in<br />
March l/L=32÷37%; in April l/L=22÷30%; in May<br />
l/L=16÷28%.<br />
The form of the leaves at the immature plant<br />
from March is predominant ovat-eliptical and at<br />
mature in May is elliptical lanceolat.<br />
The growing in length of the petiole is more<br />
pronounced in April 104÷280mm.<br />
The arch parallel nervatiune is numerical<br />
constant in all of the phases.<br />
The surface of the limb leaf mature is contained<br />
between 71,24÷145,2 cm² and the weight of the<br />
green leaves is 24,49 g/10 mature leaves.<br />
So it can be confirmed that the foliar biomass<br />
of the population of A.ursinum from Luncavita Forest<br />
is lower by comparison with the morphological<br />
studies on the same harvestes species from Botosani<br />
area ( 35,85 g/10 leafes) [8].
Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010)<br />
Table 1. Biometrical elements on wild garlic leafes<br />
Months/<br />
Leaf<br />
number<br />
m<br />
a<br />
r<br />
c<br />
h<br />
a<br />
p<br />
r<br />
i<br />
l<br />
m<br />
a<br />
y<br />
L<br />
mm<br />
l<br />
mm<br />
Lp<br />
mm<br />
l/L<br />
%<br />
R<br />
no<br />
M<br />
g<br />
1 145 50 98 34 18 0,71<br />
2 120 45 81 37 17 0,56<br />
3 150 50 100 33 19 0,8<br />
4 110 35 74 32 17 0,57<br />
5 125 44 81 35 17 0,6<br />
6 100 35 75 35 17 0,5<br />
7 148 49 100 33 18 0,75<br />
8 143 50 100 35 19 0,7<br />
9 135 45 88 33 17 0,69<br />
10 149 50 100 33 19 0,79<br />
1 200 60 256 30 22 1,48<br />
2 210 60 280 28 22 1,5<br />
3 157 35 104 22 17 1,04<br />
4 168 39 106 23 17 1,09<br />
5 205 59 280 28 22 1,4<br />
6 195 59 280 30 22 1,35<br />
7 155 35 105 22 17 1,01<br />
8 188 57 270 30 22 1,23<br />
9 190 57 268 30 22 1,26<br />
10 209 60 280 29 22 1,42<br />
1 261 70 359 26 23 2,8<br />
2 185 40 193 22 17 2,31<br />
3 250 70 360 28 23 2,62<br />
4 260 71 360 27 23 2,9<br />
5 240 67 343 27 19 2,38<br />
6 187 41 192 21 17 2,32<br />
7 180 39 195 22 17 1,9<br />
8 259 70 358 27 23 2,79<br />
9 227 37 324 16 18 2,18<br />
10 235 38 325 16 18 2,29<br />
The harvesting of the bulbs was realized in<br />
February before the entry in vegetation of the plants.<br />
In the studied area the i<strong>de</strong>ntified bulbs had different<br />
sizes (Fig.4): max.length=50mm with the diameter<br />
D=7mm; min. length =12mm with the diameter<br />
D=3mm.<br />
The number of roots is contend between 7÷10.<br />
The medium mass of the bulb is 2,4 g/piece.<br />
69<br />
Fig. 2. Bulbus with radix on wild garlic<br />
(original photo)<br />
Table 2. Biometrical elements on<br />
wild garlic bulbs<br />
Months/<br />
Bulb<br />
number<br />
f<br />
e<br />
b<br />
r<br />
u<br />
a<br />
r<br />
y<br />
L<br />
mm<br />
M<br />
g<br />
D<br />
mm<br />
No.<br />
roots<br />
1 30 3,3 5 8<br />
2 14 1,2 4 7<br />
3 25 3,1 5 7<br />
4 50 4,5 7 10<br />
5 40 3,8 7 10<br />
6 13 1,1 3 8<br />
7 20 1,3 5 7<br />
8 35 3,6 6 9<br />
9 12 1,1 3 7<br />
10 15 1,2 4 7<br />
Fig. 3. The percentage ratio-leaf finesse wild garlic<br />
Legend: S1-sample march; S2-sample april;S3sample<br />
may
Contribution to the biometrical and phytibiological.../Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010)<br />
Fig. 4. Measurements of bulbs on wild garlic<br />
The infloresecense is umbeliform arranged on a<br />
florifera strain that passes the height of the leaves.<br />
The floral stalk leaves from the same place take a<br />
vaulted form. At the base of the stalks there are<br />
bacterias wich form an involucres. The flower is type<br />
3, specific mococotyledonous, and the tricarperal<br />
ovary crushed emits a specific smell of the garlic and<br />
has a sweety taste wich attracts the bugs (Fig.5).<br />
Fig.5. Inflorescence of A. ursinum<br />
From organoleptic point of view there has been<br />
seen the next things: all of the vegetal products<br />
harvested-roots,bulbs,leaves,flowers-they have a<br />
piquant taste and powerful smell of garlic; the roots<br />
have second branches and the bulbs (white-yellow)<br />
are sourroun<strong>de</strong>d by white and transparent<br />
membranes; the green leaves on the both faces are<br />
elliptical lanceolat and the pedicels are smooth, that<br />
means the <strong>de</strong>termination ucrainicum Kleopow et<br />
Oxner [1,2].<br />
70<br />
The informations from literature of speciality<br />
about the <strong>de</strong>termination of un<strong>de</strong>rspecies of Allium<br />
ursinum are very little because of the similarities<br />
between un<strong>de</strong>rspecies ursinum and un<strong>de</strong>rspecies<br />
ucrainium. Even, the difference can be realized when<br />
the plants reach the level of inflorescence.<br />
With the help of the magnifier can be observed that<br />
the pedicels don’t prezent papillae and they have a<br />
smooth surface (Fig.6) characteristic of the<br />
un<strong>de</strong>rspecies ucrainicum [1].<br />
Fig.6. “Pediceli” and ovary “tricarpelar” of<br />
A. ursinum (original photo)<br />
The spreading of the ursinum un<strong>de</strong>rspecies,<br />
inclu<strong>de</strong>s areas from Mountains Macin -Greci,<br />
Tiganca, Niculitel- but there is not specified the area<br />
of the Luncavita Forest [2]. Also, the recent studies<br />
realized in North Dobrogea show on the<br />
Gymnospermio-Celtetum Association the presence of<br />
the Allium ursinum species but there aren’t any<br />
references about the un<strong>de</strong>rspecies [13].<br />
4. Conclusions<br />
Our studies realized in Luncavita Forest (O.S.<br />
Macin, U.P. I, “Izvorul lui Gavrila”) shows the<br />
presence of the wild garlic on large areas but only on<br />
north hills near water.<br />
The literature informations of speciality are<br />
confirmed concerning the exigency of the species<br />
against trophicity of the soil and our observations<br />
shows an affinity of the wild garlic by Corylus<br />
avellana.
Mariana Lupoae et al. / Ovidius University Annals, Biology-Ecology Series 14: 67-71 (2010)<br />
We found only an foliar dimorphism in the first<br />
fenophase in March when the report l/L is high<br />
32÷37% opposite the values from May l/L=16÷28%.<br />
The mass of the leaves is 24,49 g/10 the values of the<br />
leaves is lower comparative with the population of<br />
the wild garlic from other zones (Botosani) and the<br />
mature bulbs grow until 50mm length with a mass<br />
about 2,4g.<br />
The harvested vegetal products-bulbs,flowershave<br />
a specific smell of garlic.<br />
The fitobiological and biological analysis<br />
permitted us an i<strong>de</strong>ntify in premiere, of the<br />
subspecies studied from the Macin Mountains<br />
(Luncavita Forest) and that would be: Allium ursinum<br />
L. ssp. ucrainicum Kleopow et Oxner.<br />
The investigation of the natural population is<br />
necesarry because of the biosintetical potential<br />
therefore it can be influented by <strong>de</strong> pedoclimatic<br />
conditions from the area of the sampling of the<br />
plants.<br />
The studies un<strong>de</strong>rtaken by us can offer the<br />
premise of the harvest,conservation and processing of<br />
some vegetal products from wild garlic in or<strong>de</strong>r to<br />
improve the farmocognostic researches.<br />
5. References<br />
[1] CIOCÂRLAN V., 2000. Flora ilustrată a<br />
României–Pteridophyta et Spermatophyta,<br />
Editura Ceres, Bucureşti, pg. 919-925.<br />
[2] SĂVULESCU T., Flora Republicii Socialiste<br />
România, Editura Aca<strong>de</strong>miei Republicii Socialiste<br />
România , 1966, Vol. XI, pg.193-266.<br />
[3] TITA I., 2005. Botanica farmaceutica editia a IIa,<br />
Editura Didactica si Pedagogica Bucuresti, pg.<br />
854-863.<br />
[4 ] DJURDJEVIC L.,, Dinic A., Pavlovic P.,<br />
Mitrovic M., Karadzic B., Tesevic V., 2003.<br />
Allelopathic potential of Allium ursinum L.,<br />
Biochemical Systematics and Ecology 32, pg.533-<br />
544.<br />
[5] ONCEANU (LUPOAE) Mariana, Miron Tudor<br />
Lucian, Dinica Rodica. Studiul unor principii<br />
active din specia Alium ursinum recoltată din<br />
flora spontană, publicat în rezumat, Conferinţa<br />
Naţională a Societăţii Ecologice din România,<br />
Galaţi, octombrie 2009.<br />
71<br />
[6] STAJNER D., POPOVIC B.M., Canadanovic-<br />
Brunet J., Stajner M., 2008. Antioxidant and<br />
scavenger activities of Allium ursinum,<br />
Fitoterapia 79, p. 303-305.<br />
[7] ARHANA SENGUPTA et al., 2004. Allium<br />
Vegetables in Cancer Prevention: An Overview,<br />
Asian Pacific Journal of Cancer Prevention, Vol<br />
5: 237-245.<br />
[8] MIHĂILESCU R., Mitroi G. Iacob E. Miron A.,<br />
Stănescu U., Gille E. , Creţu R., Ionescu E.<br />
Giurescu C. , 2008. Obtaining of phytoproducts<br />
for the cardiovascular diseases profilaxy, Note 1<br />
Some investigation of the Allium ursinum<br />
chemical composition , The 5’th Conference on<br />
Medicinal and Aromatic Plants of Southeast<br />
European Countres , BRNO.<br />
[9] CONSTANTIN D. CHIRITA et al., 1964.<br />
Fundamentele naturalistice si metodologice <strong>ale</strong><br />
tipologiei si cartarii station<strong>ale</strong> forestiere , Editura<br />
Aca<strong>de</strong>miei RPR, pag.110-113 .<br />
[10] *** FARMACOPEA ROMANA, 1993. Ediţia<br />
a-X-a , Editura Medicală Bucureşti , pg. 10-63.<br />
[11] BUCUR L.,ISTUDOR V. et al., 2002. Analiza<br />
farmacognostica, Instrument <strong>de</strong> <strong>de</strong>terminare a<br />
i<strong>de</strong>ntitatii puritatii si calitatii produselor veget<strong>ale</strong>,<br />
Editura Ovidius University Press, Constanta, pg.<br />
7-87.<br />
[12] BERCU R., BAVARU A., 2007. Biometrical<br />
and morpho-anatomical observations on Acer<br />
monspessulanum L. (Aceraceae) leaves,<br />
Contributii Botanice, XLII, Gradina Botanica<br />
“Alexandru Borza” Cluj Napoca, pg. 105-110.<br />
[13] PETRESCU M. Cercetări privind biodiversitatea<br />
unor ecosisteme forestiere din Dobrogea <strong>de</strong> Nord,<br />
Editura Nereamia, Napocae-Tulcea, pg.61-72,<br />
2004.<br />
[14] NEGULESCU E., SAVULESCU Al., 1957.<br />
Dendrologie, Editura Agro-Silvica <strong>de</strong> Stat,<br />
Bucuresti, pg. 184-188.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
DINITROPHENYL DERIVATIVES ACTION ON WHEAT GERMINATION<br />
Cristina Amalia DUMITRAS -HUTANU*,<br />
*„Al. I. Cuza” University of Iasi, 11 Carol I,<br />
Iasi-700506, Romania, hutanu_amalia@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: Several dinitrophenyl ethers such as 2,4-dinitroanisol, 2,4-dinitrophenetol, 2,4-dinitro-1-<br />
(octa<strong>de</strong>cyloxy) benzene, 3-(2,4-dinitrophenoxy)propane-1,2-diol or other similar compounds have been<br />
synthesized and tested comparatively to some well-known metabolic inhibitors and stimulators within the<br />
germination experiments. As a result, the weight of the resulted plantlets was diminished by 2,4-dinitroanisol and<br />
3-(2,4-dinitrophenoxy)propane-1,2-diol treatments (1.15 g/lot and 32.03 mg/plantlet in the case of 2,4dinitroanisol;<br />
0.11 g/lot and 22.3 mg/plantlet in the case of 3-(2,4-dinitrophenoxy)propane-1,2-diol).<br />
Dinitrophenyl ethers inhibited seed germination, most probably by blocking oxidative phosphorylation. A novel<br />
mechanism of action of these pestici<strong>de</strong>s was discussed. Consequently,the toxicity processes of these pestici<strong>de</strong>like<br />
compounds and metabolic inhibitors was discussed in direct relationship with their infrared absorbance and<br />
fluorescence quenching.<br />
Keywords: pestici<strong>de</strong> toxicity, dinitrophenyl ethers, dintirophenols, wheat germination.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Dinitro<strong>de</strong>rivatives, especially the aromatics, are<br />
frequently used as intermediates in the manufacture<br />
of pharmaceuticals, dyes, pestici<strong>de</strong>s and explosives.<br />
They have multiple biological actions, being used as<br />
insectici<strong>de</strong>s, fungici<strong>de</strong>s, herbici<strong>de</strong>s and acarici<strong>de</strong>s [1,<br />
2]. However, Environment Protection Agency in<br />
SUA (EPA) inclu<strong>de</strong>d the dinitrophenols on the list of<br />
national priorities and in concentration of 3-46 mg<br />
dinitrophenol/kg body kill; no antidote is known<br />
(max. admissible dose 70 ppb in water, EPA, 2004).<br />
It is assumed that dinitrophenols hin<strong>de</strong>r the proton<br />
translocation through the mitochondrial inner<br />
membrane and therefore oxidative phosphorylation is<br />
inhibited (ATP is no longer formed and the cells<br />
<strong>de</strong>prive of essential energy supply). It is also possible<br />
that the dinitrophenols act toxically due to the<br />
inhibition of formation of some triplet states (instable<br />
biradicals) by a resonance process with the triplet<br />
structures in the living cells (A. Szent-Gyorgyi-Nobel<br />
Prize, 1957) [3, 4, 5, 6, 7, 8]. Because the existing<br />
data are inconclusive and do not support a precise<br />
action mechanism of dintrophenyl <strong>de</strong>rivatives on<br />
living organisms, it was necessary to synthesize some<br />
dinitrophenols and dinitrophenyl ethers whose<br />
biological activity should be tested.<br />
The purpose of this paper is to compare the<br />
biological activity of some synthetic compounds<br />
containing the di- and nitrophenyl moiety with that of<br />
some well-known metabolic inhibitors and<br />
stimulators. Because germination experiments are<br />
easy, cheap, fast and spectacular, the testing of the<br />
action of some action of some known and newly<br />
synthesized substances on living organisms will be<br />
performed using germinating cereal seeds [3-5]. The<br />
possible mechanism of toxicity of these chemicals<br />
and pestici<strong>de</strong>s are discussed in the light of the<br />
biostructural theory by Eugen Macovschi as well as<br />
the chemiosmotic theory by Peter Mitchell [6, 7, 8].<br />
2. Material and Methods<br />
Biological material. The wheat samples<br />
(Triticum aestivum), Henika variety, were taken from<br />
the Agricultural Research Station in Suceava. The<br />
1000 seeds weighed 37.2 g and had a residual<br />
humidity of 12%. Chemical reagents. The reagents<br />
used were of analytic purity (Merck, Sigma,<br />
Chimopar) and the solution and the water slurries<br />
were prepared using redistilled water. Thus,<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Dinitrophenyl <strong>de</strong>rivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010)<br />
dinitro<strong>de</strong>rivatives such as 2,4-dinitrophenetol, 2,4dinitroanisol,<br />
3-(2,4-dinitrophenoxy)propane-1,2-diol<br />
and 2,4-dinitrophenyl-glutathione were synthesized.<br />
Several solutions of dinitrophenyl ethers and<br />
dinitrophenols with the concentrations 4x10 -3 M were<br />
prepared. A blank with bidistilled water was also<br />
carried out.<br />
Equipment. The chemical syntheses were<br />
carried out using the organic chemistry lab equipment<br />
of the Chemistry Department of “Al. I. Cuza”<br />
University of Iasi. The experiments and the<br />
germination <strong>de</strong>terminations were performed in Petri<br />
dishes, on double Watmann no. 1 filter paper at room<br />
temperature. The separation and purification of the<br />
compounds obtained were carried out using thin layer<br />
chromatography on silica gel (Kieselgel 60F254,<br />
Merck) and on silica gel column. The infrared spectra<br />
were taken on a Jasco FT/IR660Plus Fourier<br />
spectrometer in the range from 0 to 15000 cm -1 .<br />
Procedure. The germination parameters were<br />
measured according to ISTA recommendations (Seed<br />
Science and Technology, 1993), however we worked<br />
also with lots of 50 seeds which were laid to<br />
germinate on filter paper, in Petri dishes, in three<br />
repetitions. The first count took place after three days<br />
(energy of germination, EG), the second after 7 days<br />
(germination rate, GR). The germinated, abnormal<br />
and <strong>de</strong>ad seeds as well as the resulting plantlets were<br />
counted.<br />
The treatment lasted for an hour, followed by<br />
the distribution of the seeds uniformly in the Petri<br />
dishes, on double filter paper, together with the<br />
treatment solution. The seeds with a visible root were<br />
consi<strong>de</strong>red germinated. The seeds were watered daily<br />
with 5 ml of redistilled water. The plantlets were cut<br />
at the level of the seeds 7 days after, measured and<br />
weighed (height, H, in cm and mass, m, in grams).<br />
Statistics. The results were processed using the<br />
Tukey test [9]. The mean square <strong>de</strong>viation sx of the<br />
samples was also calculated, as well as t factor, with<br />
a view to compare the results obtained un<strong>de</strong>r the<br />
action of different treatments.<br />
3. Results and Discussion<br />
As for the stimulative effect, the most active<br />
substance in these experiments proved to be<br />
resorcinol, which increased slightly by 5.5% and<br />
74<br />
phenylalanine by 6.3% the average mass of plantlets<br />
as compared to the blank. 2,4-Dinitrophenol inhibited<br />
total the germination process of wheat seeds, (Table<br />
1).<br />
1 2 3 4 5 6<br />
Fig. 1. The biological effect of some nitrophenyl<br />
<strong>de</strong>rivatives and other compounds on wheat<br />
germination. 1 – Blank (water); 2 – DNP; 3 – DNG;<br />
4 – DNA; 5 – resorcinol; 6 – L-β-phenylalanine.<br />
Table 1. The toxicity of 2,4-dinitrophenol (DNP), 3-<br />
(2,4-dinitrophenoxy)propane-1,2-diol (DNG), 2,4dinitroanisol<br />
(DNA), resorcinol and L-βphenylalanine<br />
and at concentrations 4x10 -3 M in a<br />
wheat seeds germination experiment.<br />
Treatment *)<br />
1 - Blank<br />
(water)<br />
2 - DNP,<br />
4x10 -3 M<br />
3 –DNG,<br />
4x10 -3 M<br />
4 – DNA,<br />
4x10 -3 M<br />
5 –<br />
resorcinol,<br />
4x10 -3 M<br />
Germination<br />
Rate<br />
(G. R.)<br />
Plantlets<br />
size<br />
(S, cm)<br />
Average<br />
of roots<br />
mass<br />
(m, mg)<br />
90% 6.4+0.6 18.7+1.3<br />
0% 0 0<br />
42% 2.5+0.6 8.6+3.2<br />
85% 4.6+0.6 13.9+0.1<br />
83% 5.6+0.7 18.9+0.2<br />
6 – L-βphenylalanine,<br />
4x10 -3 M<br />
87% 6.0+0.8 19.1+0.6<br />
D (Tukey test) 4.3 1.3 1.2<br />
The height of the plantles treated with 2,4dinitrophenol<br />
(DNP), 3-(2,4-dinitrophenoxy)propane-1,2-diol<br />
(DNG), 2,4-dinitroanisol (DNA),<br />
resorcinol and L-β-phenylalanine and at
Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010)<br />
concentrations 4x10 -3 M, as compared to blank<br />
(water) treatment. It is apparent from the table that<br />
the root mass treatments with resorcinol and L-βphenylalanine<br />
is higher than the control samples.<br />
Number of<br />
plantlets<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
Blank<br />
0<br />
40 60 80 100 120<br />
Plantlets size (mm)<br />
Lot A<br />
Lot B<br />
Lot C<br />
Average<br />
Fig. 2. The height of the plantlets of the lots blanck<br />
Number of<br />
platlets<br />
3<br />
2<br />
1<br />
DNG<br />
0<br />
20 30 40 50 60 70<br />
Plantlets size (mm)<br />
Lot A<br />
Lot B<br />
Lot C<br />
Average<br />
Fig. 3 . The height of the plantlets of the lots treated<br />
with 3-(2,4-dinitrophenoxy)propane-1,2-diol)<br />
75<br />
Number of<br />
plantlets<br />
10<br />
8<br />
6<br />
4<br />
2<br />
DNA<br />
0<br />
40 60 80<br />
Plantlets size (mm)<br />
Lot A<br />
Lot B<br />
Lot C<br />
Average<br />
Fig. 4 – The height of the plantlets of the lots treated with<br />
2,4-dinitroanisol.<br />
Number of<br />
plantlets<br />
10<br />
8<br />
6<br />
4<br />
2<br />
Resorcinol<br />
Lot A<br />
Lot B<br />
Lot C<br />
Average<br />
0<br />
40 60 80 100<br />
Plantlets size (mm)<br />
Fig. 5. The height of the plantlets of the lots treated<br />
with resorcinol.
Dinitrophenyl <strong>de</strong>rivates action on wheat germination / Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010)<br />
Number of<br />
plantlets<br />
Fig. 6. The height of the plantlets of the lots treated<br />
with L-β-phenylalanine<br />
Of all the five figures can be seen as only for<br />
seedlings lengths blank if the three lots are very close.<br />
In other cases the range of lengths of seedlings was<br />
higher. These differences show once again disrupting<br />
the <strong>de</strong>velopment of seedlings in the presence of<br />
chemicals, whether stimulatory or inhibitory effect.<br />
Toxicity<br />
16<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
L-β-phenylalanine<br />
2.4-Dinitrophenol<br />
Lot A<br />
Lot B<br />
Lot C<br />
Average<br />
40 60 80 100<br />
Plantlets size (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 2 4 6 8 10<br />
Concentration (mM)<br />
Fig. 7 – The biological effect of 2,4-dinitrophenol on<br />
wheat germination.<br />
Toxicity seen in this figure is that of 2,4dinitrophenol<br />
(DNP).<br />
76<br />
At concentrations of 3x10 -3 M, DNP does not<br />
allow the germination of wheat seeds<br />
The toxicity mechanism of these pestici<strong>de</strong>s,<br />
pestici<strong>de</strong>-like compounds and metabolic inhibitors<br />
may be discussed in direct relationship to their<br />
infrared absorbance and fluorescence quenching (not<br />
shown). Thus, all of them have a significant<br />
absorbance at about 6000 cm -1 in IR, corresponding<br />
to ∆G of ATP formation (as previously shown by G.<br />
Drochioiu, personal communication) and quench the<br />
fluorescence of tryptophan and other biological<br />
compounds. Fluorescence quenching of tryptophan<br />
(1µg/µl) was tested using 2,4-dinitro-ortho-cresol<br />
(DNOC). The intense quenching activity of DNOC<br />
was associated with a stronger uncoupling property.<br />
According to Drochioiu’s hypothesis, one must<br />
take into consi<strong>de</strong>ration the fact that the pH<br />
modification could be a secondary phenomenon,<br />
being possible to transfer the energy of triplet states<br />
to the ADP molecule, which incorporates it as ATP.<br />
The transition from an excited state to a normal<br />
state leads to the release or absorption of a proton,<br />
<strong>de</strong>pending on the acid or base character of the<br />
compound that is in an excited state.<br />
2,4-Dinitrophenols act as uncouplers of the<br />
breathing from oxidative phosphorylation, which<br />
results in an intensified oxygen consumption, without<br />
ATP synthesis. Dinitrophenols normally disturb ATP<br />
production within the cellular mitochondria, because<br />
the ATP is the molecule which stores and supplies<br />
energy for cellular activities [6-8]. The present<br />
theories, such as P. Mitchell’s chemiosmotic theory,<br />
claim that, unlike the other enzymes in the<br />
mitochondrial respiratory chain, the ATP pumps<br />
protons from the intermembrane space towards the<br />
matrix. Thus, the energy that the other enzymes in the<br />
chain use to accumulate protons in the intermembrane<br />
space is recuperated. This energy is necessary for the<br />
ADP phosphorylation reaction with the mineral<br />
phosphate, in the presence of Mg ions, the reaction<br />
being endothermic and requires more than 31 kJ/mol.<br />
Present research showed that it is possible for<br />
the dinitrophenyl ethers to act in a non-chemical way,<br />
probably through radical and triplet status formation,<br />
and that the proton translocation could be a<br />
secondary phenomenon in the process of oxidative<br />
phosphorylation. The action mechanism of the<br />
compounds investigated is concordant with Eugen
Cristina Amalia Dumitras - Hutanu /Ovidius University Annals, Biology-Ecology Series 14: 73-77 (2010)<br />
Macovschi’s well-known biostructural theory, but<br />
contradicts Peter Mitchell’s chemiosmotic<br />
hypothesis.<br />
4. Conclusions<br />
Development of seedlings is disrupted in the<br />
presence of chemicals, whether stimulatory or<br />
inhibitory effect.<br />
Dinitrophenyl ethers and phenolic <strong>de</strong>rivatives<br />
displayed a similar pattern of biological activity<br />
inhibiting seed germination, most probably by<br />
blocking oxidative phosphorylation. Therefore, we<br />
discussed a mechanism of biological activity as well<br />
as that of toxicity related to the energy transfer in<br />
biological systems to form ATP. The proton<br />
translocation through the biological membranes could<br />
be a secondary phenomenon, but the most important<br />
event in the toxicity process of dinitrophenyl<br />
<strong>de</strong>rivatives. Further research is still necessary to<br />
clarify the specificity of the biological activity of di-<br />
and nitrophenols.<br />
5. References<br />
[1] BEWLEY J.D., Black M., 1994 - Seeds,<br />
Physiology of <strong>de</strong>velopment and germination,<br />
Plenum press, 2nd Ed., New York and London.<br />
[2] COMĂRIŢĂ E., Şol<strong>de</strong>a C., Dumitrescu E., 1986<br />
- Chimia <strong>şi</strong> tehnologia pestici<strong>de</strong>lor, Ed. Tehnică,<br />
Bucureşti, 188 pp.<br />
[3] DUMITRAS-HUTANU, C. A., Pui, A.,<br />
Drochioiu, G., 2008 - Dinitrofenil <strong>de</strong>rivati cu<br />
posibile aplicatii in medicina si biologie:<br />
mecanisme <strong>de</strong> actiune si toxicitate, Materi<strong>ale</strong> si<br />
procese innovative. Simp. V, ZFICPM, Iasi,<br />
Editura Politehnium, 61-66.<br />
[4] DUMITRAŞ-HUŢANU C. A., Pui, A.,<br />
Gradinaru, R., and Drochioiu, G., 2008 -<br />
Toxicity of dinitrophenyl <strong>de</strong>rivatives used as<br />
pestici<strong>de</strong>s and their environmental impact,<br />
Lucrări ştiinţifice USAMV Ia<strong>şi</strong>, seria<br />
Agricultură, 51.<br />
[5] DUMITRAŞ-HUŢANU C. A., Pui A., Jurcoane<br />
S., Rusu E., Drochioiu G. 2009 - Biological<br />
effect and the toxicity mechanisms of some<br />
77<br />
ninitrophenyl ethers. Roum. Biotechnol. Lett. ,<br />
Vol. 14(6), 4893-4899 pp.<br />
[6] LEHNINGER A. L., 1987 - Biochimie, Ed.<br />
Tehnică, Bucureşti, Vol. 2, 473, 547 pp.<br />
[7] DROCHIOIU G., 2006 - In Life and mind. In<br />
search of the physical basis. S. Savva (ed.)<br />
Trafford Publ., Canada, USA, Ireland & UK, 43<br />
pp.<br />
[8] MITCHELL P., 1978 - David Keilin’s respiratory<br />
chain concept and its chemiosmotic<br />
consequences, Nobel Lecture.<br />
[9] SNEDECOR G. W., 1994 - Statistical methods<br />
applied to experiments in agriculture and<br />
biology, The Iowa Stat Univ. Press, 255 pp.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE ACTION OF SOME INSECTICIDES UPON PHYSIOLOGICAL INDICES IN<br />
RANA (PELOPHYLAX) RIDIBUNDA<br />
Alina PĂUNESCU*, Cristina Maria PONEPAL*, Octavian DRĂGHICI*, Alexandru Gabriel MARINESCU*<br />
* University of Pitesti, Faculty of Science, Departament of Ecology<br />
Târgu din V<strong>ale</strong> Street, no.1, Pitesti,410087, Romania, alina_paunescu@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: The goal of this work is to study the physiological changes induced by the action of three insectici<strong>de</strong><br />
(Carbetox, Actara 25WG and Reldan 40EC) in Rana (Pelophylax) ridibunda. The animals used in the experiment<br />
were divi<strong>de</strong>d in four experimental lots: two lots of control individuals (first lot was kept at 4-6ºC and the second<br />
lot at 22-24ºC) and two experimental lots in which the animals were treated with toxic substance and kept at 4-<br />
6ºC, respectively at 22-24ºC. The toxic was administrated with intraperitoneal shots (one shot every two days, in<br />
a scheme of three weeks). At the end of the experiment we <strong>de</strong>terminate number of erythrocytes (RBC), leukocytes<br />
(WBC) and glycemia values. We observe a <strong>de</strong>crease in number of blood cell (RBC and WBC) as well as an<br />
increase a glycemia values.<br />
Keywords: Carbetox, Actara 25WG, Reldan 40EC, frog, erythrocytes, leukocytes, glycemia<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
A number of factors have been suggested for<br />
recently observed amphibian <strong>de</strong>creases, and one<br />
potential factor is pestici<strong>de</strong> exposure. The use of<br />
pestici<strong>de</strong>s in agriculture can have effects on<br />
amphibian within or adjacent to application areas [1,<br />
2].<br />
Asi<strong>de</strong> from direct <strong>de</strong>position or drift, insectici<strong>de</strong>s<br />
can reach aquatic habitats via runoff, which <strong>de</strong>pends<br />
on precipitation, soil conditions, and slope of the<br />
catchments area [3]. The effect of insectici<strong>de</strong>s on<br />
large aquatic organisms varies with the test organism.<br />
Frogs were found to be more sensitive and may serve<br />
as a biological indicator for pestici<strong>de</strong> contamination<br />
in waterways [4].<br />
Our goal is to study the effect of three<br />
insectici<strong>de</strong>s (Carbetox, Actara 25WG and Reldan<br />
40EC) in some physiological parameters (number of<br />
erythrocytes and leukocytes, glycemia level) in Rana<br />
(Pelophylax) ridibunda at two heat level (4-6ºC and<br />
22-24ºC).<br />
Carbetox (malathion) is one of the most wi<strong>de</strong>ly<br />
used organophosphorous pestici<strong>de</strong>s with numerous<br />
agricultural and therapeutic applications, and<br />
exposure to environmentally applied malathion can<br />
lead to adverse systemic effects in anurans.<br />
Cutaneous absorption is consi<strong>de</strong>red a potentially<br />
important route of environmental exposure to<br />
organophosphorous compounds for amphibians,<br />
especially in aquatic environments [5]. It is slightly<br />
toxic via the <strong>de</strong>rmal route.<br />
Actara 25WG is a neonicotinoid insectici<strong>de</strong><br />
active against a broad range of commercially<br />
important sucking and chewing pests and it has as its<br />
component the major active ingredient, thiamethoxam<br />
(25%). Thiamethoxam's chemical structure is slightly<br />
different than the other neonicotinoid insectici<strong>de</strong>s,<br />
making it the most water soluble of this family.<br />
Reldan 40EC is an insectici<strong>de</strong> from the class of<br />
organophosphates. The active substance of this<br />
insectici<strong>de</strong> is chlorpyrifos.<br />
2. Material and Methods<br />
Adult specimens of amphibians (Rana<br />
ridibunda), of both sexes, captured in spring (April-<br />
May) from the surrounding areas of the city Pitesti<br />
(Romania) were kept unfed in freshwater aquaria.<br />
The water was changed daily to avoid the<br />
accumulation of toxic substances. After 10 days of<br />
adaptation in the lab, when they were unfed, the frogs<br />
were separated in lots, which were used separately for<br />
the following experiments: two lots of control<br />
individuals, containing animals kept in laboratory at<br />
4-6 o C, respectively at 22-24ºC with no treatment, in<br />
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running water which was changed everyday, (1) one<br />
lot containing animals which were subjected to<br />
treatment with insectici<strong>de</strong> and kept at 4-6ºC, (2) a<br />
second lot containing animals which were subjected<br />
to treatment with insectici<strong>de</strong> and kept at 22-24ºC.<br />
The toxic was administered by intraperitoneal<br />
shots, one shot every two days, in a scheme of 3<br />
weeks. The administered dosage of insectici<strong>de</strong> was<br />
not lethal as none of the subjects died through the<br />
experiment. We used three different types of<br />
insectici<strong>de</strong>: Carbetox (active substance is malathion)<br />
in a dose of 0.01ml/g body weight, Actara 25WG<br />
(active substance is thiamethoxame) in a dose of<br />
0.4mg/g body weight and Reldan 40EC (active<br />
substance is chloropyrifos-methyl) in a dose of<br />
0.01ml/g body weight.<br />
The number of erythrocytes and leukocytes was<br />
microscopically <strong>de</strong>termined with a Thoma cells<br />
numbering chamber, by using a small amount of<br />
blood collected from the heart [6]; the glycemia level<br />
has been <strong>de</strong>terminate using an Accutrend GCT.<br />
3. Results and Discussions<br />
The number of erythrocytes in the frog<br />
individuals subjected for three weeks to treatment<br />
with 0.01ml/g body weight of Carbetox was<br />
significantly affected as shown in Figure 1. The<br />
difference between the number of erythrocytes which<br />
was <strong>de</strong>termined for the control and the ‘treated’ lot at<br />
4-6ºC, an average <strong>de</strong>crease of 16.68% was found in<br />
the treated frog individuals who seemed to be related<br />
to the intense hemolytic activity. At 22-24ºC we<br />
registered a <strong>de</strong>crease with 37.01% in a number of<br />
erythrocytes.<br />
In animals treated with Actara 25WG in a dose<br />
of 0.4mg/g body weight, there has been a <strong>de</strong>crease in<br />
the number of erythrocytes with 35.11% to the<br />
control value for specimens kept at 4-6°C and with<br />
37.42% for animals treated with insectici<strong>de</strong> and kept<br />
at 22-24°C.<br />
We mention that similar results in the number of<br />
erythrocytes in the lake frog were obtained by other<br />
researchers in similar experimental conditions in fish.<br />
Thus, Ponepal [7] found a <strong>de</strong>crease in the number of<br />
erythrocytes in fish un<strong>de</strong>r the action of Actara 25WG<br />
insectici<strong>de</strong>, as well as a <strong>de</strong>crease in the oxygen<br />
consumption. Also, Dhembare [8] recor<strong>de</strong>d <strong>de</strong>creased<br />
80<br />
hemoglobin, the number of erythrocytes, leukocytes<br />
and platelets in fishes were exposed to LC50 of some<br />
insectici<strong>de</strong>s for seven days.<br />
As shown in Figure 1, as compared to the values<br />
recor<strong>de</strong>d for the control individuals of frog, the<br />
number of erythrocytes increases by 51.14% for the<br />
animals which were treated with Reldan 40EC in a<br />
dose of 0.01 ml/g of body weight and kept at 4-6ºC,<br />
while animals treated with the same concentration of<br />
Reldan 40EC but kept at 22-24ºC the number of<br />
erythrocytes increases by 76.88%. Increased number<br />
of erythrocytes un<strong>de</strong>r the action of Reldan 40EC has<br />
also been noticed by Păunescu et al [9].<br />
number of erythrocytes/ml blood<br />
1000000<br />
900000<br />
800000<br />
700000<br />
600000<br />
500000<br />
400000<br />
300000<br />
200000<br />
100000<br />
0<br />
358166.7<br />
481111.1<br />
298400<br />
225608.2<br />
232405.6<br />
301077.8<br />
851000<br />
691888.9<br />
control Carbetox Actara 25WG Reldan 40EC<br />
Fig. 1. The influence of some insectici<strong>de</strong> upon<br />
number of erythrocytes in Rana (Pelophylax)<br />
ridibunda<br />
4-6ºC<br />
22-24ºC<br />
The number of leukocytes (Fig.2) at the two<br />
heat levels registered similar changes to that of the<br />
number of red blood cells as can be seen in Figure 1.<br />
Carbetox insectici<strong>de</strong> in a dose of 0.01ml/g body<br />
weight <strong>de</strong>termined a <strong>de</strong>crease in number of<br />
leukocytes with 87.26% as compared with the witness<br />
value, at 4-6ºC. An intensive leucopenia was also<br />
registered at 22-24ºC, when the number of leukocytes<br />
<strong>de</strong>creases with 101.93%. The number of leukocytes<br />
<strong>de</strong>creases by 28.52% to the witness for animals<br />
treated with Actara 25WG and kept at 4-6°C, while<br />
the value of this in<strong>de</strong>x is lower, 62.06% as compared<br />
to the witness, at higher temperatures<br />
(22-24ºC). Reldan 40EC in a dose of 0.01 ml/g of<br />
body weight was also affected the number of<br />
leukocytes. As shown in figure 2, the difference<br />
between the number of leukocytes which was<br />
<strong>de</strong>termined for the control kept at 4-6ºC and the<br />
‘treated’ lot kept at the same temperature, an average
Alina Păunescu et al. / Ovidius University Annals, Biology-Ecology Series 14: 79-82 (2010)<br />
<strong>de</strong>crease of 53.07% was found in the treated frog<br />
individuals. Similar results were obtained at 22-24ºC<br />
when the numbers of leukocytes <strong>de</strong>crease by 68.81%<br />
of the control value. Similar effects have been carried<br />
out by [10] studying the effects of chloropyrifos on<br />
mice.<br />
number of leukocytes/ml blood<br />
700<br />
600<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
423.9444<br />
516.3333<br />
226.3889 209.9444<br />
303<br />
195.8889<br />
198.9444<br />
161<br />
control Carbetox Actara 25WG Reldan 40EC<br />
4-6ºC<br />
22-24ºC<br />
Fig. 2. The influence of some insectici<strong>de</strong> upon<br />
number of leukocytes in Rana (Pelophylax) ridibunda<br />
The glycemia level was found to be significantly<br />
influenced by Carbetox insectici<strong>de</strong>. Thus, as shown in<br />
Figure 3, at a concentration of 0.01ml/g body weight,<br />
this in<strong>de</strong>x increases after three weeks of treatment to<br />
61.29% of the control value at 4-6ºC. The same<br />
concentration of this toxic <strong>de</strong>terminate, at 22-24ºC an<br />
increase of blood glucose concentration with<br />
212.09%. It has been reported in several studies that<br />
hyperglycemia is one of the si<strong>de</strong> effects in poisoning<br />
by OP in subchronic exposure and in acute treatment<br />
[11, 12, and 13]. Several studies have <strong>de</strong>monstrated<br />
some evi<strong>de</strong>nce for damage in pancreatic exocrine<br />
function after anticholinesterase<br />
insectici<strong>de</strong> intoxication [14, 15, 16, and 17]. The<br />
stimulation of pancreatic secretion secondary to<br />
cholinergic stimulation seems to be responsible for<br />
the <strong>de</strong>velopment of pancreatitis [18, 19, 20, and 21].<br />
The influence of Actara 25WG is also felt in the<br />
glucose level, whose values are shown in Figure 3. Its<br />
analysis shows an increase of glucose by 85.07%<br />
compared to witness for animals kept at a temperature<br />
of 4-6°C and treated with a concentration of 0.4mg/g<br />
Actara 25WG and 153.05% for the animals kept at<br />
22-24°C and treated with the same concentration of<br />
toxic. Reldan 40EC in a concentration of 0.01ml/g<br />
body weight <strong>de</strong>terminate, after three weeks of<br />
treatment, an increase of glycemia level with<br />
127.41% as compared to the witness value in the case<br />
81<br />
of animals kept at 4-6ºC and with 173.59% in the<br />
case of animals kept at 22-24ºC.<br />
These changes occur due to inhibition of<br />
glucose tissue by the toxic, and inhibition of Krebs<br />
cycle and glicolise enzymes, this leading to<br />
accumulation of glucose in the blood.<br />
mg glucosis/ml blood<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
11.16667<br />
22.72222<br />
18<br />
34.83333<br />
20.66667<br />
57.5<br />
25.38889<br />
62.16667<br />
control Carbetox Actara 25WG Reldan 40EC<br />
Fig. 3. The influence of some insectici<strong>de</strong> upon<br />
glycemia in Rana (Pelophylax) ridibunda<br />
4. Conclusions<br />
4-6ºC<br />
22-24ºC<br />
Analyzing comparatively the influence of three<br />
insectici<strong>de</strong>s (Carbetox, Actara 25WG and Reldan<br />
40EC) upon some physiological indices in Rana<br />
(Pelophylax) ridibunda, we found that these <strong>de</strong>crease<br />
(in percentage) the number of erythrocytes and<br />
leukocytes and increase the glycemia values. Only<br />
Reldan 40EC insectici<strong>de</strong> causes an increase in RBC.<br />
On the other hand, the toxic effect of these<br />
insectici<strong>de</strong>s was proven to be more powerful at 22-<br />
24ºC than 4-6ºC.<br />
5. References<br />
[1] BERRILL M, BERTRAM S, PAULI B, 1997 -<br />
Effects of pestici<strong>de</strong>s on amphibian embryos and<br />
larvae. In: Green DM (ed) Amphibians in <strong>de</strong>cline:<br />
Canadian studies of a global problem. Reports<br />
from the <strong>de</strong>clining amphibian population task<br />
force. Herpetol Conserv, 1:233–245.<br />
[2] GREULICH K, HOQUE E, PFLUGMACHER S,<br />
2002 - Uptake, metabolism, and effects on<br />
<strong>de</strong>toxication enzymes of isoproturon in spawn and<br />
tadpoles of amphibians. Environ Toxicol Saf, 52:<br />
256–266.
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[3] LIESS M, SCHULZ R, LIESS MHD, ROTHER<br />
B, KREUZIG R, 1999 - Determination of<br />
insectici<strong>de</strong> contamination in agricultural<br />
headwater streams. Wat Res, 33: 239–247.<br />
[4] CALUMPANG SMF, MEDINA MJB, TEJADA<br />
AW, MEDINAJR, 1997 - Toxicity of<br />
Chlorpyrifos, Fenubucarb, Monocrotophos, and<br />
Methyl Parathion to fish and frogs after a<br />
Simulated Overflow of Paddy Water. Bull.<br />
Environ. Contam. Toxicol., 58: 909-914.<br />
[5] WILLENS S, STOSKOPF M, BAYNES R,<br />
LEWBART G, TAYLOR S, KENNEDY-<br />
STOSKOPF S, 2006 - Percutaneous malathion<br />
absorption by anuran skin in flow-through<br />
diffusion cells. Envtl. Toxicol. & Pharm, 22:<br />
263-267.<br />
[6] PICOŞ CA, NĂSTĂSESCU GH, 1988 - Lucrări<br />
practice <strong>de</strong> fiziologie animală. Tipografia<br />
Universităţii din Bucureşti, Bucureşti, 107, 122-<br />
123, 192-195.<br />
[7] PONEPAL MC, PĂUNESCU A, DRĂGHICI O,<br />
MARINESCU AlG, 2006 - Research on the<br />
changes of some physiological parameters in<br />
several fish species un<strong>de</strong>r the action of the<br />
thiametoxame insectici<strong>de</strong>. In: Proceedings 36th<br />
International Conference of IAD: 163-167.<br />
[8] DHEMBARE AJ, PONDHA GM, 2000 -<br />
Hematological changes in fish, Punctius sophore<br />
exposed to some insectici<strong>de</strong>s. Journal<br />
Experimental Zoo India, 3(1): 41-44.<br />
[9] PĂUNESCU A, PONEPAL CM, DRĂGHICI O,<br />
MARINESCU AlG, 2009 - The influence of<br />
Reldan 40EC insectici<strong>de</strong> upon physiological<br />
indices in Rana ridibunda. Lucrări Ştiinţifice<br />
USAMVB Seria B, LIII: 173-178.<br />
[10] AMBALI S, AKANBI D, IGBOKWE N,<br />
SHITTU M, KAWU M, AYO J, 2007 -<br />
Evaluation of subchronic chlorpyrifos poisoning<br />
on hematological and serum biochemical changes<br />
in mice and protective effect of vitamin C. The<br />
Journal of Toxicological Sciences, 32: 111-120.<br />
[11] GUPTA PK, 1974 - Malathion induced<br />
biochemical changes in rats. Acta Pharmacol.<br />
Toxicol., 35(3): 191–194.<br />
[12] RODRIGUES MR, PUGA FR, CHENKER E,<br />
MAZANTI MT, 1986 - Short term effect of<br />
malathion on rats’ blood glucose and on glucose<br />
82<br />
utilization by mammalian cells in vitro.<br />
Ectotoxicol. Environ. Safety, 12 (2): 110–113.<br />
[13] MATIN MA, HUSAIN K, 1987 - Cerebral<br />
glycogenolysis and glycolysis in malathiontreated<br />
hyperglycaemic animals. Biochem.<br />
Pharmacol., 36(11): 1815–1817.<br />
[14] GOKEL Y, GULALP B, ACIKALIN A, 2002 -<br />
Parotitis due to organophosphate intoxication. J.<br />
Toxicol. Clin. Toxicol. J., 40(5): 563–565.<br />
[15] PANIERI E, KRIGE JE, BORNMAN PC,<br />
LINTON DM, 1997 - Severe necrotizing<br />
pancreatitis caused by organophosphate<br />
poisoning. J.Clin. Gastrenterol., 25: 463–465.<br />
[16] DRESSEL TD, GOODALE RL, ARNESON<br />
MA, BORNER JW, 1979 - Pancreatitis as a<br />
complication of anticholinesterase insectici<strong>de</strong><br />
intoxication. Ann. Surg., 189: 199–204.<br />
[17] LANKISCH PG, MULLER CH,<br />
NIEDERSTADT H, BRAND A, 1990 - Painless<br />
acute pancreatitis subsequent to anticholinesterase<br />
insectici<strong>de</strong> (parathion) intoxication. Am. J.<br />
Gastroenterol., 85: 872–875.<br />
[18] KANDALAFT K, LIU S, MANIVEL C,<br />
BORNER JW, DRESSEL TD, SUTHERLAND<br />
DE, GOODALE RL, 1991 - Organophosphate<br />
increases the sensitivity of human exocrine<br />
pancreas to acetulcholine. Pancreas, 6: 398–403.<br />
[19] GOODALE RL, MANIVEL JC, BORNER JW,<br />
LIU S, JUDGE J, LI C, TANAKA T, 1993 -<br />
Organophosphate sensitizes the human pancreas<br />
to acinar cell injury: an ultrastructural study.<br />
Pancreas, 8: 171–175.<br />
[20] WEIZMAN Z, SOFER S, 1992 - Acute<br />
pancreatitis in children with anticholinesterase<br />
insectici<strong>de</strong> intoxication. Pediatrics, 90: 204–206.<br />
[21] MORITZ F, DROY JM, DUTHEIL G, MELKI<br />
J, BONMARCHAND G, LEROY J, 1994 - Acute<br />
pancreatitis after carbamate insectici<strong>de</strong><br />
intoxication. Intens. Care Med., 20: 49–50.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
CHANGES OF SOME PHYSIOLOGICAL PARAMETERS IN PRUSSIAN CARP<br />
UNDER THE ACTION OF SOME FUNGICIDE<br />
Maria Cristina PONEPAL, * Alina PĂUNESCU*, Alexandru Gabriel MARINESCU*, Octavian DRĂGHICI*<br />
* Universitatea din Piteşti, <strong>Facultatea</strong> <strong>de</strong> <strong>Ştiinţe</strong><br />
Str. Tg. din V<strong>ale</strong>, nr.1 Piteşti, România, e-mail: ponepal_maria@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: This study was carried out to analyze the effects of sublethal and lethal concentrations of Bravo 500<br />
SC, Champion 50 WP, Tilt 250 and Tiradin 70 PUS fungici<strong>de</strong> on some physiological parameters (oxygen<br />
consumption, breathing frequency, number of erythrocytes) of the prussian carp (Carassius auratus gibelio<br />
Bloch). The acute and subacute toxicity of fungici<strong>de</strong>s was evaluated in glass aquaria un<strong>de</strong>r semi-static conditions.<br />
Keywords: prussian carp, fungici<strong>de</strong>, Bravo, Champion, Tilt, Tiradin, breathings frequency, oxygen consumption<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The commercial product Bravo 500 SC is a<br />
concentrated suspension of chlorothalonil (500g / l);<br />
chlorothalonil (2,4,5,6 tetrachlor isophthal-nitrile) is<br />
a contact fungici<strong>de</strong> with curative and preventive<br />
action (works by stopping germination and the<br />
<strong>de</strong>velopment of spores) for combating a large number<br />
of pathogens (leaf spots, downy mil<strong>de</strong>ws,<br />
alternarioses, fruit rots, brown tor of fruit, scab) that<br />
threaten the main crops [1]. The fungici<strong>de</strong> is part of<br />
group IV of toxicity; it is not toxic to bees, warmbloo<strong>de</strong>d<br />
animals and mo<strong>de</strong>rately toxic to insects [2].<br />
Chlorothalonil and its metabolites are very toxic to<br />
fish, aquatic invertebrates and marine organisms [3]:<br />
LC50 (96 h) is of 0.25 mg/l for rainbow trout (Salmo<br />
gairdneri), 0.3 mg/l for sun perch (Lepomis<br />
macrochirus), 0.43 mg/l for sea <strong>de</strong>vil (Ictalurus<br />
punctatus), etc.<br />
Champion WP (copper hidroxi<strong>de</strong>) is a fixed<br />
copper fungici<strong>de</strong> wi<strong>de</strong>ly used for control of fungal<br />
and bacterial pathogens. Copper is highly toxic in<br />
aquatic environments and has effects in fish,<br />
invertebrates, and amphibians, with all three groups<br />
equally sensitive to chronic toxicity [4]. The<br />
Champion WP product is toxic to fish and aquatic<br />
organisms (96-hour LC50 Bluegill: 180 mg/l, 96-hour<br />
LC50 Rainbow trout: 0.023 mg/l and 48-hour EC50<br />
Daphnia: 0.065 mg/l).<br />
Tilt 250 (the active ingredient is propiconazole<br />
– triazole fungici<strong>de</strong>) has protective, curative and<br />
systemic activity. Propiconazole's mo<strong>de</strong> of action is<br />
<strong>de</strong>methylation of C-14 during ergosterol biosynthesis,<br />
and leading to accumulation of C-14 methyl sterols.<br />
The biosynthesis of these ergosterols is critical to the<br />
formation of cell walls of fungi [5]. The<br />
propiconazole is non toxic for bees, invertebrates and<br />
soil bacteriae, but is dangerous for fish and ather<br />
aquatic organisms (LC50 values ppm for freshwater<br />
fish species: bluegill 1.3-10.2, brown trout 3.5,<br />
rainbow trout 0.9-13.2, carp 6.8-21.0, catfish 2.0-5.1<br />
and fathead minnow 7.6) [6] , [7].<br />
Tiradin fungici<strong>de</strong> (the active substance is the<br />
thiuram - tetramethylthiuram disulphi<strong>de</strong> TMTD) is a<br />
general use contact fungici<strong>de</strong> with protective action,<br />
third group of toxicity. Dithiocarbamates form a large<br />
group of chemicals that have numerous uses in<br />
agriculture and medicine [8]. It is used to control<br />
Botrytis on fruit and vegetables and in seed<br />
treatment. The 96-hour EC50 for algae growth<br />
inhibition is approximately 1 mg/l (1 ppm), the 48hour<br />
EC50 for Daphnia is less than 0.21 ppm and the<br />
96-hour LC50 for fish is approximately 0.1 ppm<br />
(Bluegill sunfish, 0.0445 mg/l, Rainbow trout, 0.128<br />
mg/l and 4 mg/l carp) [9].<br />
This study was carried out to analyze the effects<br />
of sublethal and lethal concentrations – of some<br />
fungici<strong>de</strong>: Bravo 500 SC (from 0.078125 x 10 -3 to<br />
12.5 x 10 -3 ml/l water), Champion 50 WP (from<br />
0.003 to 3 mg/l water), Tilt 250 (from 0.25 to 4 ml/l<br />
water) and Tiradin 70 PUS (from 0.01 to 0.16 ml/l<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010)<br />
water) on some physiological parameters (oxygen<br />
consumption, breathing frequency, number of<br />
erythrocytes 0.078125 x 10 -3 and 1.5625 x 10 -3 ml<br />
Bravo/l water, 0.003 mg Champion/l water and 1 ml<br />
Tiradin/l water) of the prussian carp (Carassius<br />
auratus gibelio Bloch).<br />
The acute and subacute toxicity of this<br />
fungici<strong>de</strong> was evaluated in glass aquaria un<strong>de</strong>r semistatic<br />
conditions.<br />
2. Material and Methods<br />
Determinations were ma<strong>de</strong> between January<br />
2004 and October 2009 on prussian carp samples<br />
(Carassius auratus gibelio Bloch), captured from the<br />
surrounding rivers of Piteşti. Animals were<br />
acclimatized for 10 days before the completion of<br />
experiments in aquariums with a capacity of 100 l<br />
and 50 l [10], un<strong>de</strong>r conditions of natural<br />
photoperiodism, a period in which they were fed once<br />
a day (ad libitum), at around 10 am.<br />
After acclimatization in the laboratory, the fish<br />
were separated in two experimental variants (lots of<br />
10-20 fish - average weight 18 g) subjected to<br />
fungici<strong>de</strong>s.<br />
I. Determinations of oxygen consumption and<br />
frequency of respiratory movements at intervals of<br />
24, 48, 72, 96, 168 and 336 hours on all samples of<br />
these lots (<strong>de</strong>pending on survival) on prussian carp<br />
subjected to:<br />
- I.1. Bravo 500 SC in concentrations of<br />
0.00078125, 0.0015625, 0.003125, 0.0625, 0.0125<br />
ml /l water and the control lot<br />
- I.2. Champion 50 WP in concentrations of<br />
0.003, 0.03, 0.3 and 3 mg/l water and the control lot<br />
Tilt 250 in concentrations of 0.25, 0.5, 1, 2, 4 ml /l<br />
water and the control lot<br />
- I.3. Tiradin 70 PUS in concentrations of 0.01,<br />
0.02, 0.04, 0.08, 0.16 ml /l water and the control lot<br />
II. Hematological <strong>de</strong>terminations (after one,<br />
respectively two weeks of exposure to the fungici<strong>de</strong>,<br />
the fishs were sacrificed to achieve intakes of blood<br />
necessary to hematological calculations (number of<br />
erythrocytes).<br />
84<br />
II.1 – fish subjected to Bravo 500 SC in<br />
concentrations of 0.00078125, 0.0015625, ml /l<br />
water and the control lot<br />
II.2 – fish subjected to Champion 50 WP in<br />
concentrations of 0.003 mg/l water and the control lot<br />
II.3 - fish subjected to Tilt 250 in concentration<br />
1ml /l water and the control lot<br />
The fungici<strong>de</strong>s concentrations were <strong>de</strong>termined<br />
by preliminary tests of survival. The introduction of<br />
fish in solutions was done after their mixing and<br />
aeration for 5 minutes. The water temperature was<br />
16-18°C, the "immersion" solution was changed<br />
every 24 hours, and aeration of water was continuous;<br />
the fish were not fed during experiments to avoid<br />
further intervention of this factor [10]. The testing<br />
method was systematic with refreshing solution at 24<br />
hours after the calculations of the day, in aquariums<br />
of 100 l (50 l, respectively) for each experimental lot.<br />
Determination of oxygen consumption was<br />
done by means of the oximetre and Winkler method<br />
and erythrocytes were counted with Thoma chamber,<br />
using a small amount of blood from the caudal artery<br />
on the optic microscope [10], [11]. The statistical<br />
interpretation of the results was performed with<br />
ANOVA (LSD) test.<br />
3. Results and Discussions<br />
The first four figures (fig.1-4) shows the<br />
average frequency of the respiratory movements of<br />
prussian carps exposed to the action of some<br />
fungici<strong>de</strong> (Bravo, Champion, Tilt and Tiradin).
Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010)<br />
Fig.1. The influence of Bravo fungici<strong>de</strong> upon<br />
breathing frequency on prussian carp<br />
Fig.2. The influence of Champion fungici<strong>de</strong> upon<br />
breathing frequency on prussian carp<br />
Fig.3. The influence of Tilt fungici<strong>de</strong> upon<br />
breathing frequency on prussian carp<br />
85<br />
Fig.4. The influence of Tiradin fungici<strong>de</strong> upon<br />
breathing frequency on prussian carp<br />
Bravo and Champion have changed the<br />
respiratory rhythm of prussian carps in all<br />
investigated concentrations. For all concentrations<br />
tested the effect of the fungici<strong>de</strong> is initially<br />
stimulating and inhibitory as regards the frequency of<br />
respiratory movements. In two experimental variants<br />
(0.01 and 0.02 ml/l water)Tiradin is stimulating of the<br />
breathing frequency of fish; at the concentration of<br />
0.04, 0.08 and 0.16 ml/l water, the fungici<strong>de</strong> caused<br />
a <strong>de</strong>crease in the respiratory rhythm of prussian carps.<br />
Changes of prussian carps oxygen consumption<br />
exposed to the action of Bravo, Champion, Tilt and<br />
Tiradin fungici<strong>de</strong>s in differrent concentrations are<br />
shown in fig. 5-8.<br />
Fig.5. The influence of the Bravo fungici<strong>de</strong> upon<br />
oxygen consumption on prussian carp
Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010)<br />
Fig.6. The influence of the Champion fungici<strong>de</strong><br />
upon oxygen consumption on prussian carp<br />
Fig.7. The influence of the Tilt fungici<strong>de</strong> upon<br />
oxygen consumption on prussian carp<br />
Fig.8. The influence of the Tiradin fungici<strong>de</strong><br />
upon oxygen consumption on prussian carp<br />
86<br />
Clinical symptoms observed during fungici<strong>de</strong><br />
exposure (Bravo, Champion, Tilt and Tiradin) of<br />
prussian carp, correspond to observations by other<br />
authors reporting on the toxicity of fungici<strong>de</strong>s [12],<br />
[13], [14].<br />
Common symptoms of initial acute exposure to<br />
fungici<strong>de</strong>s have apparent fish hypoxia, disoriented<br />
(ataxic) at the surface, and mucus-producing effects.<br />
The oxygen consumption was found to be<br />
significantly influenced by the concentration of the<br />
used fungici<strong>de</strong>s.<br />
Bravo 500 SC ,in concentrations of 0.78125 x<br />
10 -3 , 1.5625 x 10 -3 , 3.125 x 10 -3 , 6.25 x 10 -3 and 12,5<br />
x 10 -3 ml / l Bravo, had an overall stimulating effect<br />
on oxygen consumption of prussian carps in the first<br />
phase (with variable duration: 24-96 hours after<br />
exposure) followed by restoration of energy<br />
metabolism after 7 days of exposure to toxic. Tiradin<br />
and Tilt have an inhibitory effect on the energy<br />
metabolism of prussian carps. After 7 days of<br />
exposure to Tilt, for all lots of fish tested, oxygen<br />
consumption values fall below the value recor<strong>de</strong>d<br />
before the introduction of fish in experiments.<br />
Decreased oxygen consumption un<strong>de</strong>r the action<br />
of some pestici<strong>de</strong>s and changes in respiratory rate<br />
(Dithane M 45, Reldan, Tilt,) has also been noticed<br />
by Marinescu [12] and Ponepal [13], [14].<br />
Figure 9 show the changes in the average values<br />
of erythrocytes after one and two weeks of exposure<br />
to some fungici<strong>de</strong>s.<br />
Fig. 9. The influence of some fungici<strong>de</strong> upon<br />
number of erythrocytes on prussian carp
Maria Cristina Ponepal et al./ Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010)<br />
Champion 0.003 1 1 1 9 9 9<br />
After 7 and 14 days of exposure to three<br />
0 0 0<br />
fungici<strong>de</strong> (Bravo, Champion and Tiradin) we found<br />
0.03 1 1 1 9 8 8<br />
out a significant <strong>de</strong>crease in the number of<br />
0 0 0<br />
erythrocytes. Similarly results were obtained in carp<br />
0.3 1 1 1 9 8 7<br />
by Hughes [15] after a brief exposure to<br />
0 0 0<br />
Methadathion. The <strong>de</strong>crease in RBC after 7 days<br />
3 1 1 9 8 6 4<br />
exposure to some pestici<strong>de</strong>s in fish was observed by<br />
0 0<br />
Dhembare and Pondha [16], Ponepal et al. [13],<br />
Contr 1 1 1 1 10 9<br />
[14].<br />
ol lot 0 0 0 0<br />
The fungici<strong>de</strong> Tilt, in concentration of 1 ml/l Tilt 0.25 1 1 1 1 10 10<br />
water has an stimulatory effect of erythocytes<br />
0 0 0 0<br />
number.<br />
0.5 1 1 1 1 9 9<br />
In experimental variants with Tiradin and Tilt<br />
0 0 0 0<br />
have only been observed three stages of the<br />
1 1 1 1 9 9 8<br />
sympthomatologicycal scheme <strong>de</strong>scribed by<br />
0 0 0<br />
Schäperclaus for the intoxicated fish [10].<br />
2 8 6 3 2 0 0<br />
Neurotoxic effects in rats from thiram exposure<br />
4 7 4 0 0 0 0<br />
has been noticed by Lee and Peters [17].<br />
Table 1 shows the data on fish mortality during<br />
the experiments.<br />
Chlorothalonil toxicity is lower than that<br />
indicated in the literature [2], [3], which is due both<br />
to the testing method (semi-static) and the fact that no<br />
pure chemical product has been used.<br />
Tiradin<br />
Contr<br />
ol lot<br />
0.01<br />
0.02<br />
0.04<br />
0.08<br />
0.16<br />
1<br />
0<br />
10<br />
10<br />
10<br />
10<br />
8<br />
1<br />
0<br />
10<br />
10<br />
10<br />
10<br />
7<br />
1<br />
0<br />
10<br />
10<br />
10<br />
8<br />
6<br />
1<br />
0<br />
10<br />
10<br />
10<br />
8<br />
4<br />
10<br />
10<br />
10<br />
8<br />
6<br />
1<br />
9<br />
10<br />
10<br />
6<br />
5<br />
1<br />
Contr 10 10 10 10 10 10<br />
Table 1. Lethal effect of some fungici<strong>de</strong> on<br />
prussian carp<br />
ol lot<br />
Experimental<br />
variants<br />
(fungici<strong>de</strong><br />
Conc<br />
entrat<br />
ion -<br />
ml/l,<br />
mg/l<br />
Bravo 0.000<br />
7812<br />
5<br />
0.001<br />
5625<br />
0.003<br />
125<br />
0.006<br />
25<br />
0.012<br />
25<br />
Contr<br />
ol lot<br />
The number of living<br />
specimens<br />
Immersion time (hours)<br />
24 48 72 96 168 33<br />
6<br />
10 10 10 10 10 10<br />
10 10 10 10 9 9<br />
10 10 9 9 8 7<br />
10 10 9 9 8 6<br />
10 10 9 8 7 2<br />
10 10 10 10 10 10<br />
87<br />
4. Conclusions<br />
The fungici<strong>de</strong>s investigated (Bravo, Champion,<br />
Tilt and Tiradin) have changed the respiratory rhythm<br />
of prussian carps. For all concentrations tested the<br />
effect of Bravo and Tilt fungici<strong>de</strong> is initially<br />
stimulating and inhibitory as regards the frequency of<br />
respiratory movements. In two experimental variants<br />
(0.01 and 0.02 ml/l water) Tiradin is stimulating of<br />
the breathing frequency of fish; at the concentration<br />
of 0.04, 0.08 and 0.16 ml/l water, the fungici<strong>de</strong><br />
caused a <strong>de</strong>crease in the respiratory rhythm of<br />
prussian carps.<br />
The fungici<strong>de</strong> Bravo, had an overall stimulating<br />
effect on oxygen consumption of prussian carps in the<br />
first phase followed by restoration of energy<br />
metabolism after 7 days of exposure to toxic.<br />
The fungici<strong>de</strong> Champion, un<strong>de</strong>r the<br />
concentrations of 0.003 and 3 mg/l water, had, after
Changes of some physiological parameters… / Ovidius University Annals, Biology-Ecology Series 14: 83-88 (2010)<br />
96 hours of exposure, a stimulatory effect on oxygen<br />
consumption for the prussian carp.<br />
The other two fungici<strong>de</strong>s tested (Tiradin and Tilt)<br />
have an inhibitory effect on oxygen consumption for<br />
the prussian carps.<br />
After seven and 14 days of exposure to Bravo<br />
500 SC (0.078125 x 10 -3 to 12.5 x 10 -3 ml/l water)<br />
and Champion (0,003 mg/l water) at 16-18 ºC we<br />
found out a significant <strong>de</strong>crease in the number of<br />
erythrocytes of prusian carp. Tilt 250, in<br />
concentration of 1 ml/l water causess a increase in the<br />
prussian carps erythrocytes (after 7 and 14 days of<br />
exposure).<br />
5. References<br />
[1] http://extoxnet.orst.edu/pips/chloroth.htm<br />
[2] KIDD H and JAMES DR, 1991 - Eds. The<br />
Agrochemicals Handbook, Third Edition. Royal<br />
Society of Chemistry Information Services,<br />
Cambridge, UK, (as updated). 6-10<br />
[3] DAVIES PE AND WHITE RWG, 1985 - The<br />
toxicology and metabolism of chlorothalonil in<br />
fish. 1. Lethal levels for Salmo gairdneri, Galaxias<br />
maculatus, G. truttaceus and G. auratus and the<br />
fate of super(14)C-TCIN in S. gairdneri , Aquatic<br />
Toxicology, 7 (1-2). pp. 93-105.<br />
[4] HORNE MT and DUNSON WA, 1995 - Effects<br />
of low pH, metals, and water hardness on larval<br />
amphibians, Archives of Environmental<br />
Contamination and Toxicology, 29:500-505<br />
[5] THOMSON WT, 1997- Agricultural Chemicals.<br />
Book IV: Fungici<strong>de</strong>s. 12th edition, Thomson<br />
Publications, Fresno, CA<br />
[6] http://www.epa.gov/ngispgm3/iris/irisdat<br />
[7] http://www3.bae.ncsu.edu/info1/courses<br />
[8] HOWARD PH, 1989 - Pestici<strong>de</strong>s. In : Handbook<br />
of Environmental Fate and Exposure Data for<br />
Organic Chemicals, Lewis Publishers, Chelsea,<br />
MI, pp.4-20<br />
[9] www.epa.gov/HPV/pubs/summaries<br />
[10] PICOS CA, NASTASESCU GH, 1988 - Lucrări<br />
practice <strong>de</strong> fiziologie animală. Tipografia<br />
Universităţii din Bucureşti, p.107, 122-123, 192-<br />
195.<br />
[11] ŞERBAN M, CIMPEANU G, IONESCU<br />
EMANUELA, 1993 - Meto<strong>de</strong> <strong>de</strong> laborator în<br />
88<br />
biochimia animală, Editura Didactică <strong>şi</strong><br />
Pedagogică, Bucureşti, 252 pp.<br />
[12] MARINESCU AG, DRĂGHICI O, PONEPAL<br />
C, PĂUNESCU A, 2004 - The influence of<br />
fungici<strong>de</strong> (Dithane M-45) on some physiological<br />
indices in the prussian carp (Carassius auratus<br />
gibelio Bloch), International Association for<br />
Danube Research, Novi Sad, 35: 209-214<br />
[13] PONEPAL MC, PĂUNESCU A, MARINESCU<br />
AG., DRĂGHICI O, 2009 - Effect of the<br />
Fungici<strong>de</strong> Chlorothalonil (Bravo) on Some<br />
Physiological Parameters in Prussian Carp,<br />
Lucrări ştiinţifice USAMV Ia<strong>şi</strong>, seria<br />
Horticultură, vol 52.<br />
[14] PONEPAL M., PĂUNESCU A, MARINESCU<br />
AG, DRĂGHICI O, 2009 - The Changes of Some<br />
Physiological Parameters in Prussian Carp Un<strong>de</strong>r<br />
The Action of the Tilt Fungici<strong>de</strong>, Bulletin<br />
UASVM, Cluj, 2009, 66.<br />
[15] HUGHES G., SZEGLETES T, NEMCSOK KJ.<br />
1995 - Haematological and biological changes in<br />
the blood of carp (Cyprinus carpio) following<br />
brief exposure to an organophosphoric insectici<strong>de</strong><br />
(Methidathion),Abs.Int.Biond.Symp.Cesze<br />
Bu<strong>de</strong>jovice, May<br />
[16] DHEMBARE AJ, PONDHA GM, 2000 -<br />
Haematological changes in fish. Punctius sophore<br />
exposed to some insectici<strong>de</strong>s, J.Expt. Zoo. India,<br />
3(1), 41-44.<br />
[17] LEE CC and PETERS PJ, 1967 - Neurotoxicity<br />
and behaviour effects of thiuram in rats, . Envir.<br />
Health Perspectives, 17:35-43.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
CYTOGENETIC EFFECTS INDUCED BY MANGANESE AND LEAD<br />
MICROELEMENTS ON GERMINATION AT TRITICUM AESTIVUM L.<br />
Elena DOROFTEI 1 , Maria Mihaela ANTOFIE 2 , Daciana SAVA 1 , Marioara TRANDAFIRESCU 1<br />
1 Faculty of Natural and Agricultural Science, „Ovidius” University, Constantza, University Street No. 1, Bilding<br />
B, Campus, 900552, Romania, email: edoroftei2000@yahoo.ca<br />
2 Faculty of Agricultural Sciences, Food Industry and Nature Potection, University “Lucian Blaga”from Sibiu<br />
__________________________________________________________________________________________<br />
Abstract: Our study is about the effects of manganese and lead microelements treatment on germination at<br />
Triticum aestivum L. The cytogenetic effects were studied by the calculation of the mitotic in<strong>de</strong>x, by the study of<br />
the interphase and chromosomal aberrations on the mitotic cells. We used MnSO4 and Pb(NO3)2 solutions with<br />
different concentrations: 0.0001, 0.005, and 0.01%. The Triticum seeds were preliminary imbued in water, and<br />
then they were treated for 6 and 24 hours in these solutions. The control group was treated with water. We<br />
prepared five cytological sli<strong>de</strong>s, for each sli<strong>de</strong> we have studied 10 microscopic fields with good <strong>de</strong>nsity of cells<br />
for the mitotic in<strong>de</strong>x and another 10 different microscopic fields for abnormal interphases and chromosomal<br />
aberrations. In the analyzed meristematic cells we observed an almost totally inhibition of cell division and the<br />
mitotic in<strong>de</strong>x was smaller in comparison with the control variant. The study of the frequency of the cells in<br />
different phases of the mitotic division showed that the highest percent was registered by prophases, followed at<br />
distance by telophases. We can conclu<strong>de</strong> that the heavy metals Mn and Pb have a significant mutagenic activity<br />
in vivo upon the radicles of Triticum aestivum L.<br />
Keywords: Triticum aestivum, cytogenetic effects, lead, manganese, mitotic in<strong>de</strong>x, chromosomal aberrations.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Asi<strong>de</strong> pestici<strong>de</strong>s - the most important „stress<br />
indicators” which are especially used in agriculture,<br />
other very important indicators are heavy metals.<br />
The residual waters resulting from the galvanic<br />
industry contain a real “hurricane” of heavy metals<br />
such as: mercury, cadmium, zinc, copper, lead and<br />
chrome. Generally the water pollution sources for<br />
heavy metals are as following: galvanic industry,<br />
mining, metallurgy and car industry. Copper water<br />
pollution is especially due to viticulture as the<br />
copper sulphate is used for pests’ control.<br />
Lead is eliminated mostly as a result of<br />
burning gasoline, petrol and different dyes,<br />
affecting the central nervous system in humans,<br />
creating behaviour problems and convulsions, at<br />
higher levels being lethal. Lead is spearing no<br />
organ or system being the first incriminated in<br />
boosting or getting worse a series of diseases<br />
through diminishing the body resistance. Lead<br />
effects are usually irreversible.<br />
Manganese is a nutritionally essential<br />
chemical element but also in certain conditions it<br />
can be potentially toxic. Manganese name is<br />
originating from Greek language meaning “magic”<br />
and this feature is still a<strong>de</strong>quate because the<br />
scientists are still working to un<strong>de</strong>rstand different<br />
effects of its <strong>de</strong>ficiency and toxicity effects for<br />
living organisms. However, without doubt in high<br />
levels manganese is highly toxic causing a series of<br />
pathologies based on reactive oxygen species<br />
(ROS) generation.<br />
Long term oxidative stress consequences in<br />
human where associated to the different diseases<br />
pathogenesis and toxicities namely atherosclerosis,<br />
diabetes, chronically inflammatory diseases,<br />
neurological disturbances and cardiovascudiseases.<br />
Manganese induces the oxidative stress in a<br />
time and concentration <strong>de</strong>pending manner,<br />
according to the cytotoxic parameters<br />
measurements, lactate <strong>de</strong>hydrogenase and lipid<br />
peroxidation. Also, manganese may accumulate<br />
into the cell causing cytotoxic effects and cell<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
<strong>de</strong>struction. Following different activity enzyme<br />
alteration and the alteration of gene expression the<br />
intracellular disruptions caused by manganese<br />
inclu<strong>de</strong> DNA helix broken up, chromosomes<br />
<strong>de</strong>struction and lipid peroxidation (Brooks, 1994).<br />
Our research focused in <strong>de</strong>tecting the<br />
mutagenic effects induced by heavy metals such as<br />
manganese (Mn) and lead (Pb) on higher plants<br />
using the cytogenetic analysis in Triticum aestivum<br />
L. as plant indicator for heavy metals polluting<br />
<strong>de</strong>gree in crops.<br />
The toxicity symptoms induced by heavy<br />
metals in plants are the results of some negative<br />
effects on physiological processes including:<br />
respiration and photosynthesis inhibition, water –<br />
plant relationship disruption, <strong>de</strong>creasing<br />
plasm<strong>ale</strong>ma permeability in root cells, adverse<br />
effects on the metabolic enzymes (Arduini, 1994;<br />
Chardonneres et al., 1999; Ouzounidou, 1994;<br />
Vangronsveld and Clijsters, 1994; Vennitt and<br />
Parry, 1984).<br />
2. Materials and Methods<br />
The chemical effects on chromosomes are<br />
often studied on plant material such as root tips as<br />
they are easily produced through seed germination,<br />
the experiments may be conducted all over the year<br />
and are not costly (Bateman,1977).<br />
For studying the heavy metal effect on mitosis<br />
we used solutions of MnSO4 and Pb(NO3)2 in<br />
different concentrations (0,0001%; 0,005% and<br />
0,01%) in which were submersed Triticum seeds<br />
for 6 and 24 hours, in Petri disches. As control it<br />
was used tap water. Fragments of young roots were<br />
fixed into a mixture solution of ethylic alcohol and<br />
glacial acetic acid in a volumetric rapport of 3:1 for<br />
16 h in refrigerator followed by a gentle acidic<br />
hydrolysis in HCl 1N solution for 5 min at 60°C.<br />
The roots are coloured through the Feulgen method<br />
using the Schiff reactive for 90 min followed by a<br />
water bath for 20 min. The sli<strong>de</strong>s were prepared<br />
applying squashing method and the samples were<br />
analyzed in light microscopy for the cytogenetic<br />
effects of heavy metals by calculating the mitotic<br />
in<strong>de</strong>x and revealing the chromosomal aberrations<br />
for different mitotic stages (Doroftei et al., 2008). A<br />
90<br />
Novex Holland digit camera was used for taking<br />
photographs.<br />
Table 1. Heavy metal concentrations and durations<br />
used for Triticum aestivum seeds treatments<br />
Heavy<br />
metal<br />
MnSO4<br />
MnSO4<br />
Pb(NO3)2<br />
Pb(NO3)2<br />
Concentration Variant<br />
name<br />
0,0001% V1<br />
0,005% V2<br />
0,01% V3<br />
0,0001%<br />
0,005%<br />
0,01%<br />
0,0001%<br />
0,005%<br />
0,01%<br />
0,0001%<br />
0,005%<br />
0,01%<br />
In this study 5 sli<strong>de</strong>s per variant were analyzed<br />
and for each sli<strong>de</strong> 10 microscopically filed were<br />
used for mitotix in<strong>de</strong>x calculation and for<br />
chromosomal aberrations study.<br />
3. Results and Discussions<br />
V4<br />
V5<br />
V6<br />
V7<br />
V8<br />
V9<br />
V10<br />
V11<br />
V12<br />
Treatment<br />
duration<br />
6 hours<br />
24 hours<br />
6 hours<br />
24 hours<br />
Analyzing the control untreated roots it was<br />
reve<strong>ale</strong>d the normal feature of the chromosomes<br />
and also normal cell division behaviour with a<br />
mitotic in<strong>de</strong>x of 15 %. Roots <strong>de</strong>velopment was<br />
lower when the Triticum seeds were immersed into<br />
the tested solutions, macroscopically differences<br />
being observed compared to the control. Thus, the<br />
treated roots were smaller and in a low number<br />
compared to the control.<br />
The mitotic in<strong>de</strong>x significantly <strong>de</strong>creased<br />
especially in the case of 0.01% and 24 h treatment<br />
duration for manganese and lead too, supporting the<br />
i<strong>de</strong>a that cell division is slower progressing<br />
compared to the control (Tab. 2). These<br />
microscopically observations are supported by<br />
those macroscopically (root number and size).<br />
The chromosomal aberrations are relatively<br />
diverse, being <strong>ale</strong>atory distributed and <strong>de</strong>pending
Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
on manganese and lead concentration and treatment<br />
period.<br />
For a treatment with MnSO4 solution in<br />
concentration of 0.01% for 24 h treatment, it was<br />
observed cells with big nuclei and unorganized and<br />
vacuolated features. The un-organizing process is<br />
due probably to some disequilibrium occurred as a<br />
consequence of genetic material accumulation in a<br />
too big quantity. The treatment with MnSO4<br />
solution in concentration of 0.01% for 6 h treatment<br />
it was observed that the majority of cells were in<br />
interphase and prophase and after 24 h of treatment<br />
cell plasmolysis occurred for non dividing cells.<br />
The treatment with Pb(NO3)2 solution in<br />
concentration of 0,0001% and 0,005%, for 6 or 24<br />
h treatments, induced a <strong>de</strong>crease in mitotic division<br />
frequency. For a treatment with Pb(NO3)2 of<br />
0,01%, for 24 h a significant <strong>de</strong>crease cells in<br />
mitotic division frequency it was registered as a<br />
result of summing the effects of high concentration<br />
and long period of treatment. For this later variant,<br />
nuclei unregulated in shape and size were observed<br />
and chromosome appeared either big with a relaxed<br />
chromatin either small but presenting a compact<br />
chromatin and unregulated shape. For lead too, for<br />
a concentration of 0.01% for 6 h there were<br />
observed predominantly cells in interphase or<br />
prophase and after 24 h of treatment the cell<br />
plasmolysis occurred in the non-dividing cells.<br />
The studied heavy metals solutions may have<br />
according to our results the following negative<br />
effects:<br />
1. slowing down the cell division rate (figs.<br />
1-16)<br />
2. frequent cell <strong>de</strong>gradation appearance (figs.<br />
7,8,9,10,11,13,14 )<br />
3. <strong>de</strong>hydration effect at cell level frequently<br />
inducing cell plasmolysis, more drastically<br />
at 24 h treatment (figs. 9,10,11,15,16,17)<br />
4. heterochromatinization during prophase<br />
(figs. 6,7,8,13,14)<br />
5. changes in the nuclei shape becoming<br />
elongated (figs. 6,7,8)<br />
91<br />
6. <strong>de</strong>gradation of the nucleic material in the<br />
completed <strong>de</strong>stroyed cells (figs.<br />
6,7,8,13,14)<br />
7. an early chloroplasts formatting can be<br />
observed (figs. 9,10,12,15,16,17).<br />
In all variants, comparing to the control, a<br />
<strong>de</strong>creasing in the mitotic in<strong>de</strong>x was observed (figs.<br />
1, 2, 3, 4). We recor<strong>de</strong>d the lack of cells in<br />
anaphase for the following variants: 3, 6 and 11.<br />
For the control as well as for the treated variants the<br />
predominance of prophase and telophases towards<br />
the metaphases and anaphases was registered (fig.<br />
5).<br />
The highest percentage of dividing cells is<br />
registered for the variant no. 1 (4%) and it is shown<br />
in tab. no. 2. The biggest number of cells in<br />
prophase (19) in variant 1 was registered (tab. 2).<br />
For metaphase the biggest number of cells was<br />
registered in variant 7 (14) followed by in variants<br />
1 and 9 (11).<br />
These data support the i<strong>de</strong>a that among heavy<br />
metals, manganese in large quantities impe<strong>de</strong>s the<br />
normal roots growth for higher plants as a<br />
consequence of cell division negative effects<br />
induced for the meristematic cells in Triticum<br />
aestivum.<br />
Faze <strong>de</strong> diviziune %<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Profaze<br />
Metafaze<br />
Anafaze<br />
Telofaze<br />
Fig. 1. Mitotic division phase’s frequency in<br />
Triticum aestivum treated with MnSO4 for 6 hours.
Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
Faze <strong>de</strong> diviziune %<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Fig. 2. Mitotic division phase’s frequency in<br />
Triticum aestivum treated with MnSO4 for 24<br />
hours.<br />
Faze <strong>de</strong> diviziune %<br />
Fig.3. Mitotic division phase’s frequency in<br />
Triticum aestivum treated with Pb(NO3)2 for 6<br />
hours.<br />
Faze <strong>de</strong> diviziune %<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
M t V i t V i t V i t<br />
Fig.4. Mitotic division phase’s frequency in<br />
Triticum aestivum treated with Pb(NO3)2 for 24<br />
hours.<br />
Profaze<br />
Metafaze<br />
Anafaze<br />
Telofaze<br />
Profaze<br />
Metafaze<br />
Anafaze<br />
Telofaze<br />
Profaze<br />
Metafaze<br />
Anafaze<br />
Telofaze<br />
92<br />
Fig.5. Root meristematic cells in control of<br />
Triticum aestivum. It can be observed cells in<br />
prophase, metaphase, anaphase and telophase and<br />
cytochinesis (200x).<br />
Fig.6. Root meristematic cells in Triticum aestivum<br />
treated with MnSO4 0.0001% for 6 h. It can be<br />
observed cells without content and cells in prophase<br />
and telophase. The nuclei are hypertrophic with<br />
obviously hetero-chromatinisations and<br />
vacuolisations (150x).
Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
Fig.7. Root meristematic cells in Triticum aestivum<br />
treated with MnSO4 0.0001% for 6 h. It can be<br />
observed cells without content and cells in prophase<br />
and cytochinesis. The nuclei are hypertrophic with<br />
obviously hetero-chromatinisations and<br />
vacuolisations (150x).<br />
Fig.8. Root meristematic cells in Triticum aestivum<br />
treated with MnSO4 0.005% for 6 h. It can be<br />
observed plasmolytic cells mixed with cells in<br />
prophase and telophase abnormal disposed and<br />
containing hypertrophic nuclei with obvious heterochromatinisations<br />
(150X).<br />
93<br />
Fig.9. Root meristematic cells in Triticum aestivum<br />
treated with MnSO4 0.01% for 6 h. It can be<br />
observed abnormal disposed cells without content<br />
mixed with plasmolytic cells in witch we can<br />
observed an early chloroplasts formatting (600X).<br />
Fig.10. Root meristematic cells in Triticum<br />
aestivum treated with MnSO4 0.0001% for 24 h. It<br />
can be observed long cells disposed in lines; cells<br />
are plasmolytic, during prophase and in witch we<br />
can observed an early chloroplasts formatting<br />
(600X).
Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
Fig.11. Root meristematic cells in Triticum<br />
aestivum treated with MnSO4 0.005 % for 24 h. It<br />
can be observed long cells disposed in lines; cells<br />
are plasmolytic, during prophase (600X).<br />
Fig.12. Root meristematic cells in Triticum<br />
aestivum treated with MnSO4 0.01% for 24 h. It<br />
can be observed long cells disposed in lines; cells<br />
are strongly plasmolytic, during prophase and in<br />
witch we can observed an early chloroplasts<br />
formatting (600X).<br />
94<br />
Fig.13. Root meristematic cells in Triticum<br />
aestivum treated with Pb(NO3)2 0.0001 % for 6 h.<br />
It can be observed cells without content alternating<br />
with cells in prophase and telophase, having nuclei<br />
hypertrophic (400X).<br />
Fig.14. Root meristematic cells in Triticum<br />
aestivum treated with Pb(NO3)2 0.005 % for 6 h. It<br />
can be observed cells without content alternating<br />
with cells in prophase and telophase, having nuclei<br />
hypertrophic (400X).
Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
Fig.15. Root meristematic cells in Triticum<br />
aestivum treated with Pb(NO3)2 0.01 % for 6 h. It<br />
can be observed long cells disposed in lines; cells<br />
are plasmolytic, during prophase and in witch we<br />
can observed an early chloroplasts formatting<br />
(600X).<br />
Fig.16. Root meristematic cells in Triticum<br />
aestivum treated with Pb(NO3)2 0.005 % for 24 h.<br />
It can be observed long cells disposed in lines; cells<br />
are plasmolytic, during prophase and in witch we<br />
can observed an early chloroplasts formatting and<br />
curly cell’ s walls (600X).<br />
95<br />
Fig.17. Root meristematic cells in Triticum<br />
aestivum treated with Pb(NO3)2 0.01 % for 24 h. It<br />
can be observed long cells disposed in lines; cells<br />
are plasmolytic, during prophase and in witch we<br />
can observed an early chloroplasts formatting and<br />
curly cell’ s walls (600X).<br />
4. Conclusions<br />
Based on the results of this study we may<br />
conclu<strong>de</strong> that:<br />
The heavy metals solutions used in this<br />
experiment have a great mutagenic effect on the<br />
root meristematic cells of Triticum aestivum<br />
After the heavy metals solution treatment a<br />
<strong>de</strong>crease in cell division in rate was recor<strong>de</strong>d<br />
The heavy metals have a <strong>de</strong>hydration effect at<br />
cellular level<br />
In all variants a <strong>de</strong>crease in the mitotic in<strong>de</strong>x<br />
compared to the control was observed<br />
The mutagenic effects <strong>de</strong>pends on the used<br />
heavy metals in the treatment and the treatment<br />
duration<br />
In the treated cells an early chloroplasts<br />
formatting can be observed.<br />
Cytogenetic tests on Triticum aestivum reveal a<br />
<strong>de</strong>crease in mitotic in<strong>de</strong>x after the treatment with<br />
the heavy metals solutions. These results reve<strong>ale</strong>d<br />
that the studied heavy metals present a significant
Cytogenetic effects induced by manganese... / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
mutagenic activity. The inhibition of mitotic<br />
division in the root apex induces the root growth<br />
inhibition as an active reaction of the plant when<br />
plants are exposed to the action of heavy metals in<br />
soil. Heavy metal effects are more profound but<br />
they may become visible using further molecular<br />
techniques.<br />
These results are sufficient serious arguments<br />
in the elaboration of prophylactic methods for<br />
pollution combating of surface land water,<br />
un<strong>de</strong>rground water as well as for grounding the<br />
protection measures for ecosystem maintaining.<br />
5. References<br />
[1] BROOKS, R.R., 1994: Plants and<br />
hyperaccumulate heavy metals. In: Plant and<br />
chemical elements, 88-105. Edited by M.E.<br />
Farago. Ed. VCH, Weinheim, New York, Basel,<br />
Cambridge, Tokio.<br />
[2] ARDUINI, I., GOLDBOLD, D.L., ONNIS, A.,<br />
1994: Cadmium and cooper change root growth<br />
and morphology at Pinus pinea and Pinus<br />
pinaster seedling. Physiol. Plant., 92, 675-680.<br />
[3] CHARDONNERES, A.N., BOOKUM, W.M.,<br />
VELLINGE, S., SCHAT, H., VERKLEIJ,<br />
J.A.C., ERNST, W.H.O., 1999: Allocation<br />
patterns of zinc and cadmium in heavy metal<br />
96<br />
tolerant and sensitive Silene vulgaris. J. Plant.<br />
Physiol., 155(6), 778-787.<br />
[4] OUZOUNIDOU, G., 1994: Cooper induces<br />
changes on growth, metal content and<br />
photosynthetic function of Alysum montaneum<br />
L. plants, Environ. Exp. Bot., 34, (2), 165-172.<br />
[5] VANGRONSVELD, J., CLIJSTERS, H., 1994:<br />
Toxic effects of metals. In: Plants and the<br />
chemical elements, 150-177. Edited by M.E.<br />
Farago. Ed. VCH, Weinheim, New York, Basel,<br />
Cambridge, Tokio.<br />
[6] VENNITT, S., PARRY, J.M., 1984:<br />
Mutagenicity testing: a practical approach. Ed.<br />
IRL Press, Oxford, Washington DC.<br />
[7] BATEMAN, A.J., 1977: Handbook of<br />
mutagenicity - Test procedures. Edited by B.J.<br />
Kilbey, M. Legator, W. Nichols, C. Ramel. Ed.<br />
Elsevier, North Holland, Amsterdam.<br />
[8] DOROFTEI, E., MIRON, L., ROTARU-<br />
STĂNCIC, M., 2008: Efectul mutagen al<br />
met<strong>ale</strong>lor grele cupru <strong>şi</strong> cadmiu la Allium cepa<br />
L. (The mutagenic effect of heavy metals<br />
cooper and cadmium at Allium cepa L.) În:<br />
Ar<strong>de</strong>lean, A., Crăciun, C. (eds), An<strong>ale</strong>le<br />
Societãţii Naţion<strong>ale</strong> <strong>de</strong> Biologie Celularã, XIII,<br />
225-229, Risoprint, Cluj-Napoca.
Elena Doroftei et al. / Ovidius University Annals, Biology-Ecology Series 14: 89-97 (2010)<br />
Table 2. Number of analysed cells for citogenetic studies regarding the effects of heavy metals<br />
manganese and lead on cell division<br />
Variant Total<br />
studied<br />
cell<br />
Martor<br />
Total<br />
interphase<br />
cells<br />
Total<br />
division<br />
cells<br />
Total<br />
prophase cells<br />
97<br />
Total<br />
metaphase<br />
cells<br />
Total<br />
anaphase<br />
cells<br />
Total<br />
telophase<br />
cells<br />
Nr. Nr. % Nr. % Nr. % Nr. % Nr. % Nr. %<br />
1000 850 85 150 15 51 5,1 40 4,0 30 3,0 29 2,9<br />
V1 1000 960 96 40 4,0 19 1,9 11 1,1 6 0,6 4 0,4<br />
V2 1000 968 96,8 32 3,2 14 1,4 6 0,6 4 0,4 8 0,8<br />
V3 1000 981 98,1 19 1,9 9 0,9 4 0,4 - - 6 0,6<br />
V4 1000 980 98 20 2,0 10 1,0 5 0,5 4 0,4 1 0,1<br />
V5 1000 976 97,6 24 2,4 11 1,1 6 0,6 3 0,3 4 0,4<br />
V6 1000 975 97,5 25 2,5 12 1,2 9 0,9 - - 4 0,4<br />
V7 1000 966 96,6 34 3,4 9 0,9 14 1,4 5 0,5 6 0,6<br />
V8 1000 965 96,5 35 3,5 15 1,5 9 0,9 6 0,6 5 0,5<br />
V9 1000 966 96,6 34 3,4 12 1,2 11 1,1 8 0,8 3 0,3<br />
V10 1000 974 97,4 26 2,6 11 1,1 4 0,4 3 0,3 8 0,8<br />
V11 1000 979 97,9 2,1 2,1 12 1,2 6 0,6 - - 3 0,3<br />
V12 1000 979 97,9 2,1 2,1 11 1,1 4 0,4 1 0,1 5 0,5
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
PROBLEMS OF THE HARMONIZING ENVIRONMENTAL<br />
LEGISLATION AT THE COMPARTMENT "PISCES" IN THE<br />
REPUBLIC OF MOLDOVA<br />
Petru COCIRTA, Olesea GLIGA<br />
Institute of Ecology and Geography (Aca<strong>de</strong>my of Sciences of Moldova).<br />
Aca<strong>de</strong>my Str. No.1, Chișinău, MD-2028, Republica Moldova<br />
E-mail: pcocirta@hotmail.com, camiprim@inbox.ru<br />
__________________________________________________________________________________________<br />
Abstract: In the paper are presented some results regarding principal characteristics on the structure, qualitative<br />
and comparative analysis of the national acts with EU directives as well with EU and ISO standards. It was<br />
<strong>de</strong>monstrated the compatibility of some national legislation and normative acts with EU ones. Special attention<br />
was <strong>de</strong>dicated to the rare and endangered species of fish. It was created databases on environmental legislativenormative<br />
acts of the Republic of Moldova at the compartment “Fishes”, which shows a various and satisfactory<br />
number of acts in this domain. In the final part of the paper are presented some conclusions and proposals on the<br />
<strong>de</strong>velopment of legislation and norms regarding fish species in the Republic of Moldova in accordance with EU<br />
and international requirements.<br />
Keywords: fishes and environmental legislation and normative acts state.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
According to the Declaration of Rio <strong>de</strong> Janeiro<br />
in 1992 and Agenda 21 [1], protection of biological<br />
diversity is one of the global environmental<br />
problems, which <strong>de</strong>pends on addressing the quality of<br />
life and existence of the living organism son the<br />
earth.<br />
Development and conservation of the diversity<br />
of ichthyofauna species are of paramount importance<br />
in the management of biological diversity in the<br />
marsh and aquatic ecosystems in the Republic of<br />
Moldova [2].<br />
In the past 100 years anthropogenic pressure on<br />
aquatic and march ecosystems has changed cardinal<br />
the quantity and quality of aquatic biological<br />
diversity. In Republic of Moldova the aquatic and<br />
marsh (water areas of rivers, lakes, dam lakes, ponds)<br />
ecosystems were limited to 94,6 thousand ha (2.8%<br />
of total territory), and are unevenly distributed and<br />
characterized by a wi<strong>de</strong> variety of ecological,<br />
physical, geographical, hydrochemical,<br />
hydrobiological etc. particularities. Hydrographical<br />
network consists of three main rivers - the Danube,<br />
Dniester and Prut, as well as of 3260 rivulets and<br />
3532 lakes. Most of rives were damaged, <strong>de</strong>stroyed<br />
or channeled. Biodiversity inclu<strong>de</strong>s 160 flora and<br />
125 fauna (vertebrates) species. Hydrofauna recor<strong>de</strong>d<br />
over 2135 species, including ichthyofauna - 82<br />
species [3-5].<br />
In recent <strong>de</strong>ca<strong>de</strong>s the influence of anthropogenic<br />
factors (industrial pollution, eutrophication<br />
progressive, toxicity, reducing water flow, etc.) upon<br />
ecosystems river Dniester and Prut, small rivers in the<br />
territory of the country makes major changes in<br />
biodiversity of the hydro-biocenozes with loses the<br />
viability and biological significance of rivers into the<br />
biosphere and environment.<br />
As a consequence, fish resources, which are one<br />
of the important indicators statuses of the aquatic<br />
ecosystems, <strong>de</strong>creased sharply in majority natural<br />
water objects of the Republic of Moldova and a<br />
number of species (sterlet, barbel, zarte and others)<br />
are endangered. In the Red Book of the Republic of<br />
Moldova (Second edition, 2001) [6] it was inclu<strong>de</strong>d<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
12 species of fishes (14,6 % of total number).<br />
Acording to investigations ma<strong>de</strong> by Usatai [7], it<br />
takes place the process of replacement of valuable<br />
species with less valuable.<br />
Political and socio-economic reforms in<br />
Moldova ware conditioning the need to change of<br />
attitu<strong>de</strong>s towards use of natural resources, promoting<br />
economic and social <strong>de</strong>velopment compatible with<br />
the environment. After the 2009 parliamentary<br />
elections was conditioned the need to promote the<br />
new i<strong>de</strong>as and actions for to harmonize relations in<br />
the system “Man-Society-Nature”. In this context in<br />
the Republic of Moldova there are implemented the<br />
National Strategy „Agenda 21” [8] and a number of<br />
existing national programs: The Moldovan Village,<br />
2005-2015; Program of the stabilization and<br />
economic recovery of the Republic of Moldova for<br />
the years 2009-2011 [5], and new one: Rethink<br />
Moldova. Priorities for Medium Term Development,<br />
2010-2013 [10], which would help “<strong>de</strong> facto” to<br />
economic <strong>de</strong>velopment through solving the<br />
environmental problems and respectively to stop the<br />
pollution of the environmental and <strong>de</strong>gradation their<br />
components.<br />
Current state of water areas of the Republic of<br />
Moldova induces new provocations on the<br />
elaboration of measures to <strong>de</strong>velop the actions and<br />
current species diversity of ichthyofauna, the<br />
improvement, utilization and sustainable conservation<br />
of hydrofauna in general.<br />
In the program „Rethink Moldova. Priorities<br />
Medium Term Development” among other priority<br />
issues that require to be solving there are the<br />
approximation of legislation and normative acts to<br />
those of the European Union. Achieving these<br />
<strong>de</strong>si<strong>de</strong>rates requires needs updating of the existing<br />
legislative-normative base, elaboration new laws and<br />
regulations and/or modification of those existing,<br />
adaptation national standards and normative to those<br />
international ones and/or takeover of international<br />
standards of the ISO and EN Series, etc.<br />
This <strong>de</strong>si<strong>de</strong>rates will fully covers the domain<br />
“Ichthyofauna”, including the section “Fishery”.<br />
A comprehensive study should be carried out in full<br />
for the evaluation of legal-normative basis and for<br />
highlighting some perspective problems for to legal<br />
100<br />
ensuring of the environmental management in<br />
biodiversity conservation domain.<br />
In this work are presented analytical<br />
information on current level of legislative-normative<br />
assurance of the environmental management of<br />
ichthyofauna species diversity in the Republic of<br />
Moldova and on forming of the base of legislativenormative<br />
acts in this domain.<br />
The aim of work:<br />
- analyze of legal and normativ systems on<br />
compartment "Fishes" of UN, EU and Republic of<br />
Moldova;<br />
- assessment and completing the data base of<br />
the acts referred to the Republic of Moldova;<br />
Highlighting the problems of the legislativenormative<br />
<strong>de</strong>velopment in the Republic of Moldova<br />
on ichthyofauna domain;<br />
- elaboration of the proposals for harmonization<br />
of legislation and normative in the domain of<br />
ichthyofauna to the Strategy of Sustainable<br />
Development of the Republic of Moldova, to the<br />
respective EU and international acts;<br />
In this work are presented analytical and<br />
summary information on the current level of<br />
insurance protection activities of ichthyofauna in the<br />
Republic of Moldova and creation of the base of<br />
legislative-normative acts in referred domain.<br />
2. Material and Methods<br />
Study of information regarding legislativenormative<br />
acts was performed through analyse of the<br />
data banks, catalogs and other official materials of<br />
the international and national environmental<br />
organizations.<br />
Collecting of acts materials was effectuated in<br />
the frame of the official publications (written or<br />
electronic forms) of Secretariats of the international<br />
conventions, International Organization for<br />
Standardization, European Union and others, as well<br />
as from the Republic of Moldova - periodical<br />
publication "Monitorul Oficial a Republicii<br />
Moldova", Websites of the Parliament, Government,<br />
Ministry of Justice and Ministry of Environment and<br />
others.<br />
Given the fact that information accumulated in<br />
ihthiofauna domain will serve to comparative analysis<br />
of national acts to those international, especially to
Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
European ones and will be used to <strong>de</strong>velop concrete<br />
recommendations on the harmonization of legislation,<br />
normative and standards, it was take into account the<br />
respective international methodological<br />
recommendations [11-18] and the those of national<br />
or<strong>de</strong>r - Standards of the Republic of Moldova on the<br />
principles and methodology of standardization (SM<br />
1-0:2003, MS 1-12:2002; SM 1-20:2002, SM 1-<br />
21:2002 [18, 19]) and others.<br />
Storage of the specialized information and<br />
creation of databases of legislative-normative acts on<br />
biodiversity domein (EU Directives, International<br />
Conventions, National legislation and normative) was<br />
ma<strong>de</strong> in electronic form.<br />
Collecting and processing of information on<br />
standards and technical regulations was effectuated by<br />
using existing databases of international and national<br />
Websites and formation of a register of operative<br />
information in this domain.<br />
3. Results and Discussions<br />
In the Program "Rethink Moldova. Medium<br />
Term Development Priorities, 2010-2013”<br />
approximation of legislative and normative acts to<br />
those of the EU is among the priority issues, that need<br />
resolving operational. Collection and analysis of<br />
material un<strong>de</strong>r mentioned program has permitted to<br />
highlight the following aspects of the assessment and<br />
the need of International, European and National acts<br />
for the ecological management on "Fish"<br />
compartment in the Republic of Moldova.<br />
3.1. Assessment and training normative legislative<br />
base in "PISCES"<br />
3.1.1. International legislation<br />
Various multilateral environmental agreements<br />
or conventions have been conclu<strong>de</strong>d for nature<br />
protection in general, and for aquatic fauna in special.<br />
The European Community takes an active part in the<br />
elaboration, ratification and implementation of<br />
multilateral environmental agreements. Republic of<br />
Moldova also is a part of those conventions and ma<strong>de</strong><br />
different action in accordance with ratified<br />
conventions. The principal of them which cover also<br />
the Fish compartment are named chronologically<br />
below (see Box 1).<br />
101<br />
Box 1: Multilateral environmental agreements<br />
on nature protection<br />
• Convention on Wetlands of International<br />
Importance Especially as Waterfowl<br />
Habitat (Ramsar, 1971)<br />
• Convention on International Tra<strong>de</strong> in<br />
Endangered Species of Wild Fauna and<br />
Flora (Washington,<br />
• 1973)<br />
• Convention on Conservation of Migratory<br />
Species of Wild Animals (Bonn, 1979)<br />
• Convention on the Conservation of<br />
European Wildlife and Natural Habitats<br />
(Bern, 1979)<br />
• Convention on Biological Diversity (Rio <strong>de</strong><br />
Janeiro, 1992)<br />
• Convention on Cooperation for the<br />
Protection and Sustainable Use of the<br />
Danube River (Sofia, 1994)<br />
3.1.2. EU legislation<br />
In accordance with EU recommendations [10-<br />
14], were taken to record the majority of legislative<br />
and normative acts, which are part of the acquis of<br />
Environment and need to be transposed into national<br />
law. Environmental acquis recommen<strong>de</strong>d for<br />
harmonization of national legislation is consi<strong>de</strong>rably<br />
smaller (118 documents). As the compartment “Fish”<br />
there was highlighted the following:<br />
EU Fish Protection Legislation. Within this<br />
framework, EU Nature conservation policy is<br />
implemented by one main piece of legislation –<br />
Habitats Directive - the Council Directive<br />
92/43/EEC of 21 May 1992 on the conservation of<br />
natural habitats and of wild fauna and flora. The<br />
Directive aim to provi<strong>de</strong> protection for listed species<br />
and habitats and to create the European ‘coherent<br />
European ecological network of sites – called Natura<br />
2000 to enable the maintenance or restoration of<br />
natural habitat types and the habitats of species at<br />
favorable conservation status (Art. 3, Habitats<br />
Directive). The Habitats Directive requires Special<br />
Areas of Conservation (SACs) to be <strong>de</strong>signated for<br />
listed plant and animal species, and habitats.<br />
Together, SACs and Special Protection Areas<br />
(SPAs) from Birds Directive (Council Directive<br />
79/409/EEC of 2 April 1979 on the conservation of<br />
wild birds) make up the Natura 2000 sites. SPAs and
Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
SACs areas can overlap. The Natura 2000 network<br />
already comprises more than 20,000 sites, covering<br />
almost a fifth of the EU territory.<br />
Besi<strong>de</strong>s this directive there are further relevant pieces<br />
of EU nature protection legislation referred to fish,<br />
summarized in Box 2.<br />
Box 2: EU nature (fish) protection related<br />
legislation<br />
• Council Directive 92/43/EEC on the<br />
conservation of natural habitats and of wild<br />
fauna and flora<br />
• Council Directive 1999/22/EC relating to the<br />
keeping of wild animals in zoos<br />
• Council Regulation (EC) No. 338/97 on the<br />
protection of species of wild fauna and flora<br />
by regulating tra<strong>de</strong> therein<br />
Other EU legislation relevant to nature (fish)<br />
protection inclu<strong>de</strong>:<br />
• Environmental Impact Assessment Directive<br />
(85/337/EEC), amen<strong>de</strong>d by Council Directive<br />
97/11/EC,<br />
• Access to Environmental Information<br />
Directive (90/313/EEC),<br />
• Reporting Directive (91/692/EEC).<br />
3.1.3 Legislative-normative acts of the Republic of<br />
Moldova.<br />
On June 1, 2010 database of legislativenormative<br />
acts of Republic of Moldova in the<br />
ichthyofauna domain and inter<strong>de</strong>pen<strong>de</strong>nt ones<br />
represents an impressive set of legal materials,<br />
namely:<br />
• 6 international environmental conventions to<br />
which Moldova is party;<br />
• 13 Laws of the Republic of Moldova;<br />
• 1 Presi<strong>de</strong>ntial Decree of Republic of Moldova;<br />
• 44 acts of the Republic Moldova subordinate to<br />
laws, from wich 7 Decisions of Parliament, 35<br />
Decisions of Government, 2 Acts of the Central<br />
Environmental Authority;<br />
• 1 Concept; • 2 Strategies; • 2 State Programs.<br />
The above mentioned has highlighted the<br />
importance of databases in this domain and the need<br />
to maintain and <strong>de</strong>velop them. Analyse of the results<br />
obtained show that the <strong>de</strong>velopment of legislations<br />
102<br />
and normative in the ichthyofauna domain is<br />
satisfactory.<br />
Political and social reforms from recent years<br />
have highlighted the need to harmonize legislativenormative<br />
acts, inclusive the ichthyofauna domain, to<br />
the international requirements, which will allow<br />
fulfilling the obligations of the Republic of Moldova<br />
Government, assumed by signing the international<br />
environmental conventions and facilitating the<br />
process of integration in European Union. In this<br />
context, there is evi<strong>de</strong>nt the ten<strong>de</strong>ncy and efforts for<br />
significant changes of legislative-normative acts of<br />
the Republic of Moldova, which started in last 5-6<br />
years by applying the mechanism of their<br />
harmonizing to requirements of the international<br />
legislation and normative, and, in particular, to the<br />
European requirements, in accordance with<br />
international obligations of the country.<br />
However, we should mention that several<br />
legislative-normative acts from Republic of Moldova<br />
have prescriptive nature and contain general<br />
provisions that regulate, primarily, the relations of<br />
animal kingdom protection and conservation, the<br />
management of the state protected natural areas and<br />
others. There is poorly <strong>de</strong>veloped legislative base for<br />
protection of natural complexes, for creating a green<br />
housing (frame) and application of stringent measures<br />
for recovery of environmental condition, which have<br />
directly impacts the habitats of fish species, a special<br />
vulnerable species.<br />
In national legislation lacks the mechanism<br />
nee<strong>de</strong>d to optimal ensuring of the protection and<br />
conservation activities of natural habitats of many<br />
species of fish, as well as of communities of the<br />
aquatic plant and animals.<br />
3.1.4 Legislative issues on the conservation of rare<br />
and vulnerable species<br />
Republic of Moldova legislation covers the<br />
most part of the activities of rational use and<br />
conservation of ichthyofauna species (Law on Animal<br />
kingdom (1995), Law on State Protected Areas Fund<br />
(1998), Law on fund of fisheries, fisheries and fish<br />
culture (2006), Red book of Moldova (2001).<br />
Special attention is <strong>de</strong>voted to rare and<br />
vulnerable fish species that are protected by several<br />
legislative acts, the main ones being: the Law on<br />
State Protected Areas Fund, which inclu<strong>de</strong>s 15
Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
species, the Red Book of Moldova - 12 species. We<br />
also note the primary importance of the Berne<br />
Convention (1979) to which Moldova is part from the<br />
year 1993.<br />
Harmonize national legislation with<br />
international and European requirements impose<br />
additional measures to conserve species of<br />
ichthyofauna. Were subjected to comparative analysis<br />
some legislative acts of Republic of Moldova and<br />
those more important international (European Red<br />
List (2009) [21], Council Directive 92/43/EEC [22]<br />
and the Berne Convention [23] regarding the status<br />
and the protection state of rare and vulnerable fish<br />
species. It were analyzed the status of 21 important<br />
species of fish presents on territory of the Republic of<br />
Moldova (Table 1).<br />
Table 1. Some important fish species of the Republic<br />
of Moldova un<strong>de</strong>r comparative analysis<br />
Name of Species Acts with<br />
species found<br />
Or<strong>de</strong>r Salmoniformes<br />
1. Hucho-hucho (L) – Danube<br />
salmon or Huchen<br />
2. Salmo salar (L) -<br />
Atlantic salmon<br />
3. Umbra krameri(Walbaum) –<br />
European Mudminnow<br />
LAK, LPA, RB,<br />
ERL, CD, BC 1)<br />
ERL, CD, BC<br />
LAK, LPA, RB, BC<br />
Or<strong>de</strong>r Cipriniformes<br />
4. Rutilus frisii Nordmann – LAK, LPA, RB,<br />
Black Sea Roach<br />
ERL, CD, BC<br />
5. Leuciscus leuciscus (L) –<br />
Common Dace<br />
LAK, LPA, ERL<br />
6. Leuciscus idus (L)<br />
LAK, LPA, RB,<br />
- I<strong>de</strong> or Gol<strong>de</strong>n Orfe<br />
ERL, BC<br />
7. Vimba-vimba (L) – Zarte LPA, ERL<br />
8. Barbus barbus borysthenicus LAK, LPA, RB,<br />
(Dubowsky) – Borys<br />
ERL<br />
9. Barbus meridionalis (Petenyi LAK, LPA, RB,<br />
Heckel) – Mediterranien Barbel ERL, CD, BC<br />
10.Cobitis taenia (L) – Spined<br />
Loach<br />
CD, BC<br />
103<br />
11.Gobio albipinnatus<br />
CD, BC<br />
(Vladykov Fang) – White-finned<br />
Gudgeon<br />
12.Rho<strong>de</strong>us sericeus amarus CD, BC<br />
(Bloch) – European Bitterling<br />
Or<strong>de</strong>r Gadiformes<br />
13.Lota lota (L) - Barbot<br />
LAK, LPA, RB,<br />
ERL<br />
Or<strong>de</strong>r Perciformes<br />
14.Zingel zingel (L) – Zingel LAK, LPA, RB,<br />
ERL, CD, BC<br />
15.Zingel streber (Siebold) - LAK, LPA, RB,<br />
Sreber<br />
ERL, CD, BC<br />
Or<strong>de</strong>r Acipenseriformes<br />
16.Gimnocephalus schraetzer<br />
(L) - Schraetzer<br />
17.Huso huso (L) -<br />
European Sturgeon<br />
18.Acipenser gul<strong>de</strong>nstaedti<br />
colchilus (V.Marti) – Russian<br />
Sturgeon<br />
19.Acipenser stellatus (Pallas) -<br />
Sturry Sturgeon<br />
20.Acipenster ruthenus (L) -<br />
Sterlet<br />
21.Acipenster nudiventris<br />
(Lovetyki) – Bastard Sturgeon<br />
CD, BC<br />
LAK, LPA, RB,<br />
ERL, CD, BC<br />
LAK, LPA, RB, CD<br />
LAK, LPA, RB, CD,<br />
BC<br />
LPA, CD, BC<br />
CD<br />
Note: 1) LAK - Law on Animal Kingdom, LPA - Law on<br />
State Protected Areas Fund, RB - Red book of Moldova ,<br />
ERL - European Red List, CD - Council Drective<br />
92/43/EEC, BC - Bern Convention.<br />
Analysis <strong>de</strong>monstrates that of these above<br />
mentioned, only six species (Danube Salmon, Black<br />
Sea Roach, Mediterranean Barbel, Zingel, Streber,<br />
European Sturgeon) are covered by all legislation<br />
acts un<strong>de</strong>r review, 12 species are covered by the<br />
European Red List, 15 species - Council Directive<br />
92/43/EEC and 15 species - Bern Convention,<br />
respectively.<br />
There were i<strong>de</strong>ntified 9 species, which in<br />
accordance with requirements of Council Drective<br />
92/43/EEC falling un<strong>de</strong>r Annex II and requires the<br />
<strong>de</strong>signation of special areas of conservation, as wel as<br />
un<strong>de</strong>r the Bern Convention, ratified by the Moldovan<br />
Parliament <strong>de</strong>cision No. 1546-XII of 23. 06. 93. But<br />
5 of these species (Salmo salar, Cobitis taenia,<br />
Gobio albipinnatus, Rho<strong>de</strong>us sericeus amarus,
Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
Gimnocephalus schraetser) have no-one protected<br />
status in the Republic of Moldova. Also were<br />
i<strong>de</strong>ntified 2 species (Acipenster rutenus and<br />
Acipenster nudiventri) falling un<strong>de</strong>r Annex V of the<br />
Council Directive 92/43/EEC, but the second species<br />
has no one of any protected status in country.<br />
The comparative analysis <strong>de</strong>monstrates the<br />
need to review the rarity status of the mentioned fish<br />
species and/or their inclusion in the Red Book of<br />
Moldova, in other legislation acts and/or performing<br />
other actions to perpetuate their best. These findings<br />
are in full compliance with existing legal basis of the<br />
Republic of Moldova: Art. 9, 16, 17 and 18 of the<br />
Law on the Red Book of Moldova (No. 325-XVI<br />
from 15. 12. 2005); The Common Action Plan<br />
Republic of Moldova – European Union, 2005-2007;<br />
and The Program "Rethink of Moldova. Priorities for<br />
medium term <strong>de</strong>velopment".<br />
Comparative analysis of the state and how to<br />
protect rare and vulnerable species of fish confirmed<br />
the importance of measures taken and existing needs<br />
in Moldova in this section. Increasing vulnerability a<br />
ichthyofauna species confirmed by increasing number<br />
of introduced species in the Red Book of Moldova,<br />
the second edition of. Proposals have already been<br />
<strong>de</strong>veloped [7] introducing other four species in the<br />
next edition (III) of the Red Book. They are supposed<br />
to be: Tench - Tinca tinca (L), Spirlin - Alburnoi<strong>de</strong>s<br />
bipunctatus rossicus (Berg), Crucian Carp -<br />
Carassius Carassius (L.), Wels catfish - Silurus<br />
glanis (L.).<br />
3.2. Evaluation and formation standards database<br />
in „PISCES”<br />
In the compartment "Pisces", relative to other<br />
domains there are few sets of standards, gained<br />
worldwi<strong>de</strong> by international organizations - ISO<br />
(International Standardisation Organisation) and IEC<br />
(International Electrotechnical Commission) and at<br />
the European level - CEN (European Committee for<br />
Standardization), CENELEC (European<br />
Electrotechnical Committee for Standardization) and<br />
ETSI (European Telecommunications Standards<br />
Institute) [17.18].<br />
3.2.1. International standards<br />
In accordance with electronic information<br />
provi<strong>de</strong>d by the Organization ISO [17], in querying<br />
104<br />
for section "Fish" can be found 17 ISO standards and<br />
one in elaboration and for section "Fishing and fish<br />
breeding" another 13 ISO standards. Meanwhile at<br />
the European level [18] for the section "Fish" it was<br />
highlighted 12 EN standards, from which 8 ISO<br />
standards taken by EN (European Normatives)<br />
organization.<br />
3.2.2. Standards of the Republic of Moldova<br />
The Republic of Moldova doesn’t have in action<br />
ISO and EN standards. But in Catalogue of normative<br />
documents in standardization of the Republic of<br />
Moldova [19.20] in 65.150 section “Fishing and fish<br />
farming” were not i<strong>de</strong>ntified standards in use, and in<br />
section 67.120.30 “Fish and fish products” are in<br />
force 109 GOST standards (standards of Russia<br />
adopted as national). It is clear the need of some<br />
<strong>de</strong>cisions and activities in <strong>de</strong>veloping regulations and<br />
standards in the relevant field.<br />
4. Conclusions<br />
Republic of Moldova dispose a significant base<br />
of legislative-normative acts in ichthyofauna domain.<br />
The legal regulation of ichthyofauna conservation is<br />
in continuous <strong>de</strong>velopment, already having a solid<br />
theoretical and practical basis.<br />
In the last 4-5 years it is obvious trend and<br />
efforts for significant changing of the legislativenormative<br />
acts of the Republic of Moldova by<br />
applying of the mechanism to their harmonizing to<br />
the requirements of international legislation and<br />
normative, especially, to the European requirements.<br />
In connection with the increasing vulnerability<br />
of species further efforts are nee<strong>de</strong>d for continuous<br />
<strong>de</strong>velopment of legislative basis regarding the<br />
habitats protection of vulnerable species of fish and<br />
protection of natural complexes in general, as well as<br />
creation a ecological housing and application of<br />
stringent measures to redress the environmental<br />
status.<br />
Comparative analysis of the state and protection<br />
mo<strong>de</strong> of rare and vulnerable fish species has<br />
confirmed the importance of measures taken and<br />
existing needs in the Republic of Moldova in this<br />
domain.<br />
Additional measures are nee<strong>de</strong>d on improving<br />
the mechanism and instruments to ensure optimal
Petru Cocirta, Olisea Gliga / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
operation of the protection and conservation of many<br />
species of fish natural habitats, as well as of plant and<br />
animal aquatic communities.<br />
In Republic of Moldova the ichthyofauna<br />
domain needs to move quickly to adopt international<br />
and European standards in national practice.<br />
Given that the diversity of fish species in<br />
Moldova is in its own way, unique and, un<strong>de</strong>r current<br />
conditions of climate change, utilization and damage<br />
of the ichthyofauna species genetic fund, there is<br />
need more attention, a stricter approach and effective<br />
activities for resolution of their <strong>de</strong>velopment and<br />
conservation problems.<br />
5. References<br />
[1] Agenda 21, Rio <strong>de</strong> Janeiro, 1992.<br />
[2] Republic of Moldova. Biological Diversity<br />
Conservation National Strategy and Action Plan.<br />
(Ministry of the Environment and Territorial<br />
Development. The World Bank), Chi<strong>şi</strong>nău,<br />
Ştiinţa, 2002, 100 p.<br />
[3] Republic of Moldova, First National Report on<br />
Biological Diversity. (Ministry of the<br />
Environment and Territorial Development. The<br />
World Bank), Chi<strong>şi</strong>nău, Ştiinţa, 2000, 68 p.<br />
[4] Republic of Moldova. Third National Report on<br />
the implementation of the Convention on<br />
Biological Diversity. CBD, Chisinau, December,<br />
2005.<br />
[5]http://bsapm.moldnet.md/Text/Raportul%20III/Ra<br />
pr-03-englez.pdf - data of access 4 June 2010<br />
Republic of Moldova. State of the environment<br />
Report 2006. Ministry of Ecology and Natural<br />
Resources, Chi<strong>şi</strong>nău, 2007, 85 p.<br />
[6] Red Book of the Republic of Moldova, Second<br />
edition, Stiinta, 2001, 288p.<br />
[7] USATÂI M. “Evolution, conservation, and<br />
sustainable use of diversity of ichthyofauna in<br />
aquatic ecosystems of Republic of Moldova”.<br />
Autoreferat of dissertation for the scientific<br />
<strong>de</strong>gree of doctor Habilitatus in biological<br />
sciences. Chi<strong>şi</strong>nău, 2004, 48 p. (In Romanian)<br />
[8] National Strategy of the Sustainable Development<br />
– “Moldova 21”. Supreme Economic Council<br />
un<strong>de</strong>r Presi<strong>de</strong>nt of the Republic of Moldova,<br />
PNUD Moldova, Chi<strong>şi</strong>nău, 2000, 129 pag. (In<br />
Romanian).<br />
105<br />
[9] National program “Moldovian Village, 2005-<br />
2015; Program for stabilization and re-launch of<br />
the economy in the Republic of Moldova for years<br />
2009-2011 (In Romanian) - www.gov.md.<br />
[10] Government of Moldova. Rethink Moldova.<br />
Priorities for Medium Term Development. Report<br />
for the Consultative Group Meeting in Brussels<br />
24 March 2010<br />
http://siteresources.worldbank.org/INTMOLDOV<br />
A/Resources/Rethink-Moldova-2010-2013-Fin<strong>ale</strong>dit-110310.pdf<br />
[11] White Paper on the Preparation of the<br />
Associated Countries of Central and Eastern<br />
Europe for Integration into the Internal Market of<br />
the Union, COM(95) 163 final, 3.5.1995<br />
[12] Environmental regulatory reform in the NIS: the<br />
case of the Water sector. Twelfth meeting of the<br />
EAP Task Force, 18-19 October 2000, Almaty.<br />
http://www.oecd.org/dataoecd/23/5/2382097.pdf<br />
[13] Gui<strong>de</strong> to the approximation of the European<br />
Union Environmental Legislation, SEC (97) 1608<br />
of 25.08.1997.<br />
http://ec.europa.eu/environment/gui<strong>de</strong>/contents.ht<br />
m<br />
[14] Handbook on the implementation of ec<br />
environmental legislation.<br />
http://ec.europa.eu/environment/enlarg/handbook/<br />
handbook.htm<br />
[15] COCIRTA P., CLIPA Carolina. Ecological<br />
legislation of the Republic of Moldova: Catalogue<br />
of the documents. Chisinau, Stiinta, 2008, 65 pag.<br />
(In Romanian)<br />
[16] COCIRTA P. Environmental systems and<br />
electronic information’s in the Republic of<br />
Moldova. Aca<strong>de</strong>my of Sciences of Moldova.<br />
Institute of Ecology and Geography – Chisinau,<br />
2007. 30 pag. (In Romanian)<br />
[17]http://www.standardsinfo.net/info/livelink/fetch/2<br />
000/148478/6301438/in<strong>de</strong>x.html<br />
18. http://www.cen.eu/cen/pages/<strong>de</strong>fault.aspx<br />
[19] Catalogue of normative documents in<br />
standardization of the Republic of Moldova. Year<br />
2008. National Institute of Standardization and<br />
Metrology. Vol.1,2,3. Chisinau, 2008. (In<br />
Romanian)<br />
[20] http://www.standard.md;<br />
[21] European Red List. - www.iucnredlist/Europe<br />
[22]http://ec.europa.eu/environment/nature/legislatio<br />
n/habitatsdirective/in<strong>de</strong>x_en.htm
Problems of the harmonizing environmental… / Ovidius University Annals, Biology-Ecology Series 14: 99-105 (2010)<br />
[23]http://europa.eu/legislation_summaries/environm<br />
ent/nature_and_biodiversity/l28046_en.htm<br />
106
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
BIODIVERSITY CONSERVATION IN CONSTANŢA COUNTY<br />
Silvia TURCU*, Marcela POPOVICI**, Loreley JIANU**<br />
*Ovidius University of Constanţa, Doctoral School, Biology Domain,<br />
Mamaia Avenue, No. 124, Constanţa, 900552, Romania, sscturcu@yahoo.com<br />
** Environmental Protection Agency Constanţa, Unirii Street, No. 23, Constanţa, 900532<br />
__________________________________________________________________________________________<br />
Abstract: Nature conservation is the action taken by human society to maintain and perpetuation of species of<br />
plants and animals. Recognition of the value of biodiversity in Constanta County is done by the special<br />
protection of habitats and species for an important number of protected areas. The main instrument governing the<br />
activities taking place at the perimeter and adjacent of natural areas is management plan of protected area, in<br />
accordance with existing environmental legislation.<br />
Keywords: biodiversity conservation, protected areas, administration and custody, Constanţa County.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Nature Conservation is the action taken by<br />
human society to maintain and perpetuation of<br />
species of plants and animals [1]. In our country, to<br />
provi<strong>de</strong> special measures of protection and<br />
biodiversity conservation, was instituted a tiered<br />
system of protection, conservation and use,<br />
according to the following categories of protected<br />
areas: national interest (scientific reserves, national<br />
parks, natural monuments, nature reserves, natural<br />
parks), the international interest (natural sites of<br />
universal natural heritage, geoparks, wetlands of<br />
international importance, biosphere reserves), the<br />
community interest or Natura 2000 sites (sites of<br />
Community Importance, Special Areas of<br />
Conservation Areas Special Protection Bird) or<br />
local interest [2].<br />
2. Results and Discussions<br />
In Constanţa County, there are over 900<br />
species spermatophytes present, most of these are<br />
characteristic species of steppe and forest steppe<br />
habitats, over 200 species of vascular flora of<br />
national interest, with varying <strong>de</strong>grees of<br />
vulnerability, some of these are en<strong>de</strong>mic species<br />
[3].<br />
Fauna of Constanţa County is characterized<br />
by great wealth, represented by more than 345<br />
vertebrate taxa - 45 species of mammals, 243 birds,<br />
19 species of reptiles, 10 species of amphibians and<br />
28 species of fish - and a significant number of<br />
invertebrates [3].<br />
In Constanţa County, natural and semi-natural<br />
habitats, found in all environments (aquatic,<br />
terrestrial and subterranean), are classified into<br />
seven classes (coastal and halophilic communities,<br />
continental water, scrub and grassland, forests,<br />
marshes and wetlands, screes, rock and continental<br />
sands and agricultural land and artificial<br />
landscapes) which inclu<strong>de</strong> 58 types of natural<br />
habitat and ru<strong>de</strong>ral communities (agricultural land<br />
and artificial landscapes) [4].<br />
Thus, since 1970, a number of valuable areas<br />
in terms of biodiversity were <strong>de</strong>clared reserves by<br />
<strong>de</strong>cisions of Constanţa County People’s Council.<br />
In 2000, only two of the previously <strong>de</strong>clared<br />
protected natural areas remains areas of local<br />
interest (Table 1), for the rest of these, by Law<br />
5/2000 [5] is nationally recognized protected area<br />
status.<br />
In coming years, new laws were imposed on<br />
other areas of protected area status of national<br />
interest: Government Decision 2151/2004 [6],<br />
Government Decision 1581/2005 [7], Government<br />
Decision 1143/2007 [8] and currently totaling 36<br />
protected natural areas of national interest (Table<br />
2).<br />
Since joining the European Union in 2007,<br />
Romania has emerged as one of the nations that<br />
have a true natural heritage, with many protected<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010)<br />
areas and many species listed in Annexes of Birds<br />
and Habitats Directives. Un<strong>de</strong>r European<br />
Directives, European Council Directive 92/43 EEC<br />
[9], and Birds Directive - European Council<br />
Directive 79/409 EEC [10], countries of European<br />
Union (EU) ensures maintenance or restoration of<br />
natural habitats and wild fauna and flora of<br />
Community interest in a favorable conservation<br />
status, to help maintain biodiversity.<br />
Following the transposition of these two<br />
Directives into national law was established system<br />
of protection for 42 areas: 22 special protection<br />
areas for birds (SPA), reported by Government<br />
Decision no. 1284/2007 [11] and a number of 20<br />
sites of community importance (SCI), <strong>de</strong>clared by<br />
Or<strong>de</strong>r no. 1964/2007 [12] (Table 3 and Table 4).<br />
There is a part of the “Danube Delta” Biosphere<br />
Reserve, internationally protected area, on<br />
administrative territory of Constanţa County. This<br />
is the largest protected area in the country and has a<br />
threefold international status: Biosphere Reserve,<br />
Ramsar Site and Site of World Natural and Cultural<br />
Heritage (Table 4). “Danube Delta” Biosphere<br />
Reserve has its own administrative structure<br />
established by Law 82/1993 [13]. Management plan<br />
af this protected area was <strong>de</strong>veloped by Danube<br />
Delta “Biosphere Reserve” Administration.<br />
Techirghiol Lake became the Ramsar Site on<br />
March 23, 2006 and was classified as wetland of<br />
international importance by Government Decision<br />
no. 1586/2006 [14] (Table 4). In addition to this<br />
status, Techirghiol Lake was <strong>de</strong>clared nature<br />
reserve and Bird Protection (Natura 2000). This<br />
protected area has not been attributed in custody,<br />
but “Dobrogea-Litoral” Water Directorate, in<br />
partnership with The Romanian Ornithological<br />
Society have <strong>de</strong>veloped a management plan for<br />
Lake Techirghiol trough project<br />
LIFE04NAT/RO/000220 Improving wintering<br />
condition for Branta ruficollis at Techirghiol Lake.<br />
As can be seen from Tables 1, 2, 3, 4 and 5<br />
responsibilities for managing natural protected<br />
areas, placed un<strong>de</strong>r special protection and<br />
conservation, belong to local authorities for<br />
protected natural areas <strong>de</strong>clared by <strong>de</strong>cisions of<br />
their, to “Danube Delta” Biosphere Reserve<br />
Administration for Biosphere Reserve Danube<br />
Delta and to custodians/ administrators for natural<br />
protected areas <strong>de</strong>clared by law, by Government<br />
108<br />
<strong>de</strong>cisions or by or<strong>de</strong>r of the central public authority<br />
for environmental protection. Gaining of<br />
custody/administration of natural protected areas is<br />
in accordance with the procedure of Government<br />
Decision 1533/2007 [15]. However, within six<br />
months of the signing of custody agreement for<br />
natural protected areas, custodian must <strong>de</strong>velope<br />
regulation of protected area, which contains the<br />
rules will be respected within the protected area,<br />
and within a year to effectuate the protected area<br />
management plan, in line with regulation. The<br />
measures provi<strong>de</strong>d in management plans of<br />
protected natural areas are <strong>de</strong>veloped taking<br />
account of economic requirements, social and<br />
cultural as well as on regional and local area, but<br />
with priority for the objectives which led to the<br />
establishment of protected area.<br />
3. Conclusions<br />
Recognition of the value of biodiversity in<br />
Constanta county is done by the special care and<br />
protection of habitats and species for a number of<br />
two protected areas of county interest, 36 protected<br />
natural areas of national interest, 42 protected<br />
natural areas of interest (Natura 2000 sites): 22 of<br />
Special Protection Areas for Birds (SPAs) and 20<br />
Sites of Community Importance (SCI), two natural<br />
areas of international concern.<br />
Currently, of the 82 protected areas in the<br />
county of Constanţa 68 are administered according<br />
to law, and 14 will be assumed to custody until the<br />
end of 2010.<br />
Conservation of biodiversity is in accordance<br />
with existing environmental legislation and<br />
management plan of protected areas is the main<br />
instrument governing the activities taking place at<br />
the perimeter and adjacent natural areas.<br />
Management of protected natural areas in<br />
Constanţa County will improve by <strong>de</strong>veloping the<br />
management plans, by custodians/ administrators.<br />
4. References<br />
[1] BAVARU A. et al., 2007- Biodiversitatea <strong>şi</strong><br />
ocrotirea naturii, Editura Aca<strong>de</strong>miei Române.<br />
[2] ***Government Emergency Ordinance no.<br />
57/2007 on the regime of natural protected areas,<br />
natural habitats, flora and fauna.
Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010)<br />
[3] ***Report on the Environmental Conditions in<br />
Constanţa County in 2009.<br />
[4] DONIŢĂ N. et al. 2005, "Habitatele din<br />
Romania", Editura Tehnică <strong>şi</strong> Silvică.<br />
[5] ***Law 5/2000 approving the national spatial<br />
plan and is nationally recognized and protected<br />
area status.<br />
[6] ***Government Decision 2151/2004 on the<br />
establishment of protected area regime to new<br />
areas,<br />
[7] Government Decision 1581/2005 concerning<br />
the establishment of protected area system to new<br />
areas.<br />
[8] ***Government Decision 1143/2007<br />
concerning the establishment of new protected<br />
areas.<br />
[9] ***European Council Directive 92/43 EEC on<br />
the conservation of natural habitats and wild flora<br />
and fauna adopted on May 21, 1992.<br />
[10] ***European Council Directive 79/409 EEC<br />
on the conservation of wild birds taken on April<br />
2, 1979.<br />
109<br />
[11] ***Government Decision no. 1284/2007<br />
<strong>de</strong>claring Bird specially protected areas as part of<br />
the European ecological network Natura 2000 in<br />
Romania.<br />
[12] ***Or<strong>de</strong>r of Ministry of Environment and<br />
Sustainable Development no. 1964/2007<br />
concerning the establishment of protected area<br />
system of sites of Community importance, as part<br />
of European ecological network Natura 2000 in<br />
Romania.<br />
[13] ***LAW no 82/1993 establishing Biosphere<br />
Reserve “Danube Delta”.<br />
[14] ***Government Decision no. 1586/2006 on<br />
the classification of protected areas in the<br />
category of wetlands of international importance.<br />
[15] ***Or<strong>de</strong>r no. 1533/2008 approving the<br />
Methodology for the award of administration of<br />
natural protected areas that require the<br />
establishment of administrative structures and<br />
methodology for awarding custody of protected<br />
natural areas that do not require the creation of<br />
management structures.
Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010)<br />
Table 1. Natural Protected Areas of county interest<br />
No. Protected area Administrator<br />
1. “Arborele Corylus colurna” - natural monument Constanţa Hall<br />
2. “Pâlcul <strong>de</strong> stejari brumării” - natural monument Mangalia Hall<br />
Table 2. Natural Protected Areas of national interest<br />
No. Protected area Administrator/ Custodian<br />
1.<br />
“Acvatoriul litoral-marin Vama Veche-2 Mai”<br />
- Zoological and Botanical Reserve<br />
-<br />
2.<br />
“Canar<strong>ale</strong>le din Portul Hârşova” -<br />
Morfogeological Monument<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
3.<br />
“Cetatea Histria” Scientific Reserve -<br />
Archaeological Site part of Danube Delta<br />
“Biosphere Reserve”<br />
Danube Delta “Biosphere Reserve”<br />
Administration -Tulcea<br />
4.<br />
“Dealul Alah Bair” - Complex Nature<br />
Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
5.<br />
“Dunele marine <strong>de</strong> la Agigea” - Botanical<br />
Nature Reserve<br />
A.I. Cuza University - Ia<strong>şi</strong><br />
6.<br />
“Grindul Chituc” - Scientific Reserve part of<br />
Danube Delta “Biosphere Reserve”<br />
Danube Delta “Biosphere Reserve”<br />
Administration - Tulcea<br />
7.<br />
“Grindul Lupilor” - Scientific Reserve part of<br />
Danube Delta “Biosphere Reserve”<br />
Danube Delta “Biosphere Reserve”<br />
Administration - Tulcea<br />
8. “Gura Dobrogei” - Complex Nature Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
9. “Lacul Agigea” - Zoological Nature Reserve -<br />
10. “Lacul Bugeac” - Complex Nature Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
11. “Lacul Dunăreni” - Complex Nature Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
12. “Lacul Oltina” - Complex Nature Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
“Lacul Techirghiol” - Zoological Nature<br />
13.<br />
Reserve<br />
-<br />
14. “Lacul Ve<strong>de</strong>roasa” - Complex Nature Reserve<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
“Locul fosilifer Aliman” - P<strong>ale</strong>ontological<br />
15.<br />
Monument<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
“Locul fosilifer Cernavodă”- Geological and<br />
16.<br />
P<strong>ale</strong>ontological Monument<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
“Locul fosilifer Credinţa” - P<strong>ale</strong>ontological<br />
17.<br />
Monument<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
“Locul fosilifer Movila Banului”- Geological<br />
18.<br />
and P<strong>ale</strong>ontological Monument<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
110
Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010)<br />
“Recifii jurasici Cheia” - Geological and<br />
19.<br />
Botanical Nature Reserve<br />
“Mlaştina Hergheliei” - Complex Nature<br />
20.<br />
Reserve<br />
“Obanul Mare <strong>şi</strong> Peştera ” -<br />
21. Speleological and Morfogeological Nature<br />
Reserve<br />
22. “Pădurea Bratca” - Complex Nature Reserve<br />
“Pădurea Canaraua-Fetii”- Botanical and<br />
23.<br />
Zoological Nature Reserve<br />
“Pădurea Celea Mare - V<strong>ale</strong>a lui Ene” -<br />
24.<br />
Complex Nature Reserve<br />
25. “Pădurea Cetate” - Complex Nature Reserve<br />
“Pădurea Dumbrăveni” - Botanical and<br />
26.<br />
Zoological Nature Reserve<br />
“Pădurea Esechioi” - Botanical and Zoological<br />
27.<br />
Nature Reserve<br />
“Pădurea Fântâniţa-Murfatlar” - Botanical and<br />
28.<br />
Zoological Nature Reserve<br />
“Pădurea Hagieni” - Botanical and Zoological<br />
29.<br />
Nature Reserve<br />
30.<br />
“Pereţii calcaro<strong>şi</strong> <strong>de</strong> la Petroşani” - Geological<br />
Monument<br />
“Peştera ” - Speleological<br />
31.<br />
Monument<br />
“Peştera ” - Scientific<br />
32.<br />
Speleological Reserve<br />
“Peştera ” - Speleological<br />
33.<br />
Monument<br />
“Reciful neojurasic <strong>de</strong> la Topalu” - Geological<br />
34.<br />
and P<strong>ale</strong>ontological Monument<br />
“Corbu-Nunta<strong>şi</strong>-Histria” – Scientific Reserve<br />
35.<br />
part of Danube Delta “Biosphere Reserve”<br />
“Valu lui Traian Rezervaţie” - Archaeological<br />
36.<br />
and Botanical Nature Reserve<br />
Table 3. Special Protection Areas – for Birds (SPA)<br />
111<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
The Group of Un<strong>de</strong>rwater and Speleological<br />
Exploration - Bucharest<br />
The Group of Un<strong>de</strong>rwater and Speleological<br />
Exploration - Bucharest<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
The Group of Un<strong>de</strong>rwater and Speleological<br />
Exploration - Bucharest<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
Danube Delta “Biosphere Reserve”<br />
Administration -Tulcea<br />
No. Site Name Administrator/ Custodian<br />
1. “Aliman – Adamclisi”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
2. “Allah Bair – Capidava”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
-
Biodiversity Conservation in Constanta County/ Ovidius University Annals - Biology-Ecology Series 14: 107-113 (2010)<br />
3.<br />
“Balta Mică a Brăilei”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Brăila<br />
4.<br />
“Balta Ve<strong>de</strong>roasa”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
5.<br />
“Băneasa - Canaraua Fetei”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
6.<br />
“Canar<strong>ale</strong>le <strong>de</strong> la Hârşova”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
7.<br />
“Cheile Dobrogei”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
8. “Delta Dunării <strong>şi</strong> Complexul Razim – Danube Delta “Biosphere Reserve”<br />
Sinoie”<br />
Administration -Tulcea<br />
9.<br />
“Dumbrăveni”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
10.<br />
“Dunăre – Ostroave”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
11. “Dunărea Veche - Braţul Măcin” -<br />
12.<br />
“Lacul Bugeac”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
13.<br />
“Lacul Dunăreni”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
14.<br />
“Lacul Oltina”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
15. “Lacul Siutghiol” -<br />
16. “Lacurile Taşaul – Corbu” -<br />
17. “Lacul Techirghiol” -<br />
18.<br />
“Limanu – Herghelia”<br />
The Group of Un<strong>de</strong>rwater and Speleological<br />
Exploration - Bucharest<br />
19. “Marea Neagră” EUROLEVEL<br />
20.<br />
“Pădurea Hagieni”<br />
National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
21. “Stepa Casimcea” -<br />
22. “Stepa Saraiu – Horea” -<br />
Table 4. Sites of Community Importance (SCI)<br />
No. Site Name Administrator/ Custodian<br />
1. “Balta Mică a Brăilei” National Forest Administration ROMSILVA-<br />
Forestry Department Brăila<br />
2. “Braţul Măcin” -<br />
3. “Canar<strong>ale</strong>le Dunării” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
4. “Dealul Alah Bair” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
5. “Delta Dunării” Danube Delta “Biosphere Reserve”<br />
Administration<br />
6. “Delta Dunării - zona marină” Danube Delta “Biosphere Reserve”<br />
Administration<br />
112
Silvia Turcu et al./ Ovidius University Annals, Biology-Ecology Series 14: 107-113 (2010)<br />
7. “Dumbrăveni - V<strong>ale</strong>a Urluia - Lacul National Forest Administration ROMSILVA-<br />
Ve<strong>de</strong>roasa”<br />
Forestry Department Constanţa<br />
8. “Dunele marine <strong>de</strong> la Agigea” A.I. Cuza University Iasi<br />
9. “Fântâniţa Murfatlar” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
10. “Izvoarele sulfuroase submarine <strong>de</strong> la<br />
Mangalia”<br />
GEOECOMAR<br />
11. “Mlaştina Hergheliei - Obanul Mare <strong>şi</strong> The Group of Un<strong>de</strong>rwater and Speleological<br />
Peştera Movilei”<br />
Exploration - Bucharest<br />
12. “Pădurea Esechioi - Lacul Bugeac” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
13. “Pădurea Hagieni - Cotul Văii” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
14. “Pădurea <strong>şi</strong> V<strong>ale</strong>a Canaraua Fetii – National Forest Administration ROMSILVA-<br />
Iortmac”<br />
Forestry Department Constanţa<br />
15. “Peştera Limanu” The Group of Un<strong>de</strong>rwater and Speleological<br />
Exploration - Bucharest<br />
16. “Plaja submersă Eforie Nord - Eforie Sud” EUROLEVEL<br />
17. “Podişul Nord Dobrogean” -<br />
18. “Recifii Jurasici Cheia” National Forest Administration ROMSILVA-<br />
Forestry Department Constanţa<br />
19. “Vama Veche - 2 Mai” -<br />
20. “Zona marină <strong>de</strong> la Capul Tuzla”<br />
GEOECOMAR<br />
Table 5. Natural Protected Areas of international concern<br />
No. Protected Area Administrator<br />
1. “Lacul Techirghiol” - Ramsar Site -<br />
2.<br />
“Delta Dunării” – Biosphere Reserve, Ramsar Site,<br />
World Heritage Site Natural and Cultural<br />
Danube Delta “Biosphere<br />
Reserve” Administration -Tulcea<br />
113
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE PRESENT SITUATION OF THE NOSE HORNED VIPER POPULATIONS<br />
(VIPERA AMMODYTES MONTANDONI BOULENGER 1904)<br />
FROM DOBRUDJA (ROMANIA AND BULGARIA)<br />
Marian TUDOR<br />
Universitatea Ovidius Constanţa, <strong>Facultatea</strong> <strong>de</strong> <strong>Ştiinţe</strong> <strong>ale</strong> <strong>Naturii</strong> <strong>şi</strong> <strong>Ştiinţe</strong> <strong>Agricole</strong><br />
B-dul Mamaia, nr. 124, Constanţa, 900527, România, e-mail<br />
__________________________________________________________________________________________<br />
Abstract: Due to the <strong>de</strong>struction and <strong>de</strong>terioration of the specific habitats and the increased fragmentation of the<br />
remaining ones, the nose horned viper has lost large tracts of vital living space. In addition, road kills, direct kills<br />
and collecting by humans contribute to their <strong>de</strong>cline. I tried to estimate the present situation of the nose horned<br />
viper populations in Dobrudja, based on literature and our own field data. The main goals were: to investigate the<br />
present situation of the nose horned populations in Dobrudja; to i<strong>de</strong>ntify the most suitable habitats for Vipera<br />
ammodytes montandoni; and to locate the viable populations of this viper and current threats to the nose horned<br />
viper populations.<br />
Keywords: Dobrudja, Nose-Horned Viper, viable populations<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The study of the nose horned viper in general,<br />
and of the Dobrudja subspecies in particular, can<br />
offer both herpetologists and conservationist<br />
biologists important data due to the relatively strict<br />
habitat requirements of this herpeto-taxon (particular<br />
habitat conditions, the necessary presence of certain<br />
prey-species in the habitat etc), as well as to its<br />
vulnerability to the modifications of the specific<br />
habitats. From this point of view, it is one of the i<strong>de</strong>al<br />
species for monitoring in the protected areas, as well<br />
as in those territories to be <strong>de</strong>signated protected areas<br />
in the future. The subspecies is consi<strong>de</strong>red critically<br />
endangered (CR) in the Vertebrates Red List of<br />
Romania [1] and it is inclu<strong>de</strong>d in annex 3A of OM<br />
1198/2005 (Species of European interest in need of<br />
strict protection, critically endangered species).<br />
The populations of Vipera ammodytes<br />
montandoni are in a continuous <strong>de</strong>cline [2] due to<br />
anthropogenic causes and their need for preservation<br />
is all the more imperative as the <strong>de</strong>struction of the<br />
specific habitats has increased consi<strong>de</strong>rably over the<br />
last few years.<br />
2. Material and Methods<br />
Starting with 1995, thirty-eight locations<br />
mentioned in literature ([3], [4], [5], [6], [7], [8], [9])<br />
in the Romanian area of Dobrudja have been<br />
explored with the purpose of verifying the<br />
preservation state of the nose horned viper<br />
populations. Eight more locations have been explored<br />
for the same reason in the Bulgarian region of<br />
Dobrudja in 2008.<br />
The researches took place especially in spring<br />
and autumn, when the vipers are more active and<br />
more easily recognizable in the specific habitats [10],<br />
[11], [12], [13], [14]. The explorations used visual<br />
transects as well as the method of active search in the<br />
specific habitats. [11], [15].<br />
The capture and handling of the vipers was<br />
accomplished with the help of the herpetological<br />
hook and tongs ([16], [17]). Leather gloves were used<br />
in the case of small individuals. After i<strong>de</strong>ntification<br />
and sex <strong>de</strong>termination, each individual was released<br />
in the same place where it was captured from. Also,<br />
the roads that bor<strong>de</strong>red or intersected the explored<br />
habitats were repeatedly examined, and all the road<br />
kills were photographed and collected. The searches<br />
led many times to the discovery of individuals whose<br />
<strong>de</strong>ath was a result of the direct interaction with<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010)<br />
humans. In such cases, the vipers had usually been hit<br />
to <strong>de</strong>ath with stones or other<br />
hard objects. No instances of natural <strong>de</strong>ath were<br />
i<strong>de</strong>ntified among the <strong>de</strong>ad individuals.<br />
All the inventoried individuals in each<br />
researched habitat were quantified and the<br />
<strong>de</strong>termination of the viability <strong>de</strong>gree of the<br />
populations was attempted by means of calculating<br />
the i<strong>de</strong>ntified adult/juveniles proportion [18]. The<br />
calculation of the viability <strong>de</strong>gree also took into<br />
account the state of the habitats and particularly the<br />
level of human intervention, starting from the premise<br />
that a natural or semi-natural habitat offers much<br />
better conditions for the survival of a nose horned<br />
viper population than an anthropogenic one.<br />
3. Results and Discussions<br />
The study has ren<strong>de</strong>red evi<strong>de</strong>nt certain aspects<br />
that complete the data regarding the state of the nose<br />
horned viper populations in Dobrudja. Thus, if before<br />
our researches, it was consi<strong>de</strong>red that Vipera<br />
ammodytes montandoni has a relatively large<br />
distribution in Dobrudja [7], [19], [9], our data rather<br />
bring arguments in favor of the i<strong>de</strong>a that this<br />
subspecies currently occupies small habitats in more<br />
or less strictly <strong>de</strong>lineated areas. This aspect supports<br />
the i<strong>de</strong>a that the exchanges of individuals among<br />
populations are very poor or lack completely. This<br />
may lead in time to the reduction of the intrapopulation<br />
genetic diversity.<br />
Also, most of the habitats of nose horned viper<br />
populations in Dobrudja are intersected or bor<strong>de</strong>red<br />
by roads. Thus, it was observed that, out of a total of<br />
thirty-eight locations situated in the Romanian part of<br />
Dobrudja where populations of nose horned viper had<br />
previously been mentioned [7], only in twenty-five of<br />
them (65.8%) the presence of this herpeto-taxon<br />
could be ren<strong>de</strong>red evi<strong>de</strong>nt. Numerous monasteries<br />
and hermitages have been built over the past ten years<br />
and their presence already generates a rise in the<br />
number of direct kills in some locations where there<br />
were populations of nose horned viper such as<br />
Babadag Forest, Gura Dobrogei, Dumbraveni,<br />
Hagieni and the foot of Pricopanului Peak.<br />
The existence of this Dobrudja subspecies could<br />
no longer be evi<strong>de</strong>nced in the other thirteen locations<br />
116<br />
previously mentioned as habitats for nose horned<br />
viper populations.<br />
The study has evi<strong>de</strong>nced the fact that in<br />
Dobrudja (both the Romanian and the Bulgarian<br />
si<strong>de</strong>), the largest populations of Vipera ammodytes<br />
montandoni are situated in Dumbraveni Natural<br />
Reserve, Babadag Forest, Priopcea Hill, Macin<br />
Mountains National Park, Gura Dobrogei Natural<br />
Reserve, the ruins of Adamclisi fortress, Canaraua<br />
Fetii Natural Reserve, Rusalka, Kaliakra, Bolata<br />
Dèrè, Yaillata and Kamen Bryag. Of the total<br />
inventoried individuals in the researched areas, 14%<br />
were represented by animals whose <strong>de</strong>ath was a result<br />
of the anthropogenic impact. Among these, 67% are<br />
represented by vipers killed <strong>de</strong>liberately, most having<br />
a crushed skull, and 33% are road kills, especially in<br />
spring when these reptiles prefer to bask directly on<br />
road asphalt (figure 1).<br />
67%<br />
33%<br />
Fig. 1. The raport Road kill/Direct kill<br />
road kills<br />
direct kills<br />
The estimation of population viability in<br />
Dumbraveni Natural Reserve, Babadag Forest,<br />
Priopcea Hill, Macin Mountains National Park, Gura<br />
Dobrogei Natural Reserve, the ruins of Adamclisi<br />
fortress, Canaraua Fetii Natural Reserve, Rusalka,<br />
Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag<br />
evi<strong>de</strong>nced the fact that the number of juveniles<br />
compared to that of adults is relatively high in these<br />
areas, which could thus indicate a high viability of<br />
these populations. As a whole, the situation is<br />
graphically illustrated in figure 2.<br />
In what regards the abundance of individuals in<br />
the researched populations, it was observed that in<br />
locations such as Gura Dobrogei, Babadag,<br />
Dumbraveni, Adamclisi, Hagieni, Canaraua Fetii and<br />
Bolata Dèrè, the number of i<strong>de</strong>ntified individuals is<br />
higher (figure 3). Still, this aspect only leads to the
Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010)<br />
conclusion that these populations might be larger than<br />
the ones i<strong>de</strong>ntified and investigated. Also, this aspect<br />
must be correlated with the number of field hours<br />
spent in each location. If the number of field<br />
hours spent in the Romanian Dobrudja is<br />
approximately equal (generally over 100 hours) in<br />
each location, the number of hours spent in the<br />
locations of the Bulgarian Dobrudja is much lower<br />
(an average of 10-12 hours per location). This is why<br />
it is very likely that the number of individuals in the<br />
i<strong>de</strong>ntified populations could be much higher in the<br />
Bulgarian locations. Consi<strong>de</strong>ring the time spent in<br />
each location, the relative preservation of the<br />
habitats, as well the effort of capturing the animals,<br />
all these bring arguments in favor of this hypothesis.<br />
Otherwise, in what regards these populations in<br />
the Bulgarian si<strong>de</strong> of Dobrudja, the data collected<br />
over the 2008 research season evi<strong>de</strong>nce a relatively<br />
good preservation of the nose horned viper in the<br />
natural and semi-natural habitats. No road kills or<br />
direct kills were discovered in these areas, probably<br />
due to the fact that these habitats are located at a<br />
consi<strong>de</strong>rable distance from roads and spaces<br />
<strong>de</strong>dicated to activities with anthropogenic impact.<br />
Still, given that the data collected here were gathered<br />
over a period of only a few months, it is possible that<br />
direct kills could occur sporadically due to tourism or<br />
animal grazing [20].<br />
At the same time, we estimate that the new<br />
buildings, as well as the s<strong>ale</strong> of lands that shelter<br />
vipers to investors, will lead to the <strong>de</strong>struction of<br />
their specific habitats in Bulgaria too. In both<br />
countries, the expansion of constructions and road<br />
improvement with the purpose of easing transport but<br />
also of facilitating the access of mass tourism to wild<br />
areas, will lead to the enhancement of the<br />
anthropogenic impact in areas where it either did not<br />
exist or it was sporadic.<br />
4. Conclusions<br />
The main conclusion of the study is that<br />
Dobrudja, as biogeographical area well<br />
circumscribed and with particular characteristics<br />
compared to the other parts of Europe situated at the<br />
same latitu<strong>de</strong>, still hosts viable populations of the<br />
montandoni horned viper subspecies;<br />
117<br />
The most serious danger for the preservation of<br />
this subspecies of horned viper is represented by the<br />
<strong>de</strong>struction of habitats. Immediately after come the<br />
road kills and direct kills.<br />
The populations of Vipera ammodytes<br />
montandoni in Dobrudja are isolated one from the<br />
other, therefore we believe that there are few<br />
exchanges of individuals among them or that these<br />
exchanges lack completely in some cases, leading<br />
thus to the reduction of the intra-population genetic<br />
diversity;<br />
Our data argument for the existence of at least<br />
12 areas that shelter viable populations of nose<br />
horned vipers in Dobrodja. These areas are:<br />
Dumbraveni Natural Reserve, Babadag Forest,<br />
Priopcea Hill, Macin Mountains National Park, Gura<br />
Dobrogei Natural Reserve, the ruins of Adamclisi<br />
fortress, Canaraua Fetii Natural Reserve, Rusalka,<br />
Kaliakra, Bolata Dèrè, Yaillata and Kamen Bryag.<br />
Future studies will focus on the estimation of<br />
intra-population genetic diversity and on the<br />
dynamics of certain populations of this subspecies in<br />
or<strong>de</strong>r to propose the best measures inten<strong>de</strong>d for the<br />
preservation of the Dobrudja nose horned viper.<br />
Acknowledgements<br />
This study was partly possible thanks to the<br />
UNDP/GEF Atlas Project no. 047111 “The<br />
strengthening of the national system of protected<br />
areas in Romania through the best management<br />
practices in the Macin Mountains National Park.”<br />
The researches in the Bulgarian area of<br />
Dobrudja were possible thanks to the PHARE CBC<br />
2005 Romania-Bulgaria Program RO 2005/017-<br />
535.01.02.02 “Comparative studies regarding the<br />
biodiversity of coastal habitats, the anthropogenic<br />
impact and the possibilities for the conservation of<br />
important European habitats between Cape Midia<br />
(Romania) and Cape Kaliakra (Bulgaria).<br />
We are in<strong>de</strong>bted to:<br />
Dr. Dan Cogălniceanu and Dr. Marius Skolka<br />
for providing references, valuable advice and<br />
logistics.<br />
Dr. Zsolt Török and Dr. Paul Szekely for<br />
support and references.<br />
Dr. Olivia Chirobocea for the revising of the<br />
text and accurate English translation.
The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010)<br />
5. References<br />
[1] IFTIME, A. (2005) - Reptile. In: Cartea Roșie a<br />
vertebratelor României, 173–196.<br />
BOTNARIUC ,N. & TATOLE, V. (Eds.).<br />
Bucuresti: ed. Curtea Veche.[in Romanian].<br />
[2] GIBBONS, J.W. et. al. 2000 - The Global<br />
Decline of Reptiles, Déjà Vu Amphibians<br />
BioScience, Vol 50, No.8, 653-666.<br />
[3] TOROK, Z.1999 - Note privind distribuția<br />
spațiala a herpetofaunei în zona Culmii<br />
Pricopanului, Acta oecologica, 6, 57-62.<br />
[4] TOROK, Z. 2000 - Șerpii veninoși din România<br />
(Venomous snake of Romania), Petarda, no.6,<br />
Tulcea, Aves.<br />
[5] SOS, T., 2005 - Note preliminare privind<br />
distribuția spațiala a herpetofaunei <strong>de</strong> pe<br />
Culmea Pricopanului din Parcul Național Munții<br />
Măcin, Migrans, Târgu Mureș, 7(3), 8-10.<br />
[6] OTEL, V., 1997 - Investigatii herpetologice în<br />
zona munților Măcin și podișul Babadagului,<br />
Anal Șt. IDD, 1997, 71-77.<br />
[7] FUHN, I.E. & VANCEA, St. 1961 - Reptilia<br />
(Țestoase, Șopirle, Șerpi). In: Fauna RPR.Vol.<br />
14(2). Bucuresti: Edit. Aca<strong>de</strong>miei RPR. 338 [in<br />
Romanian].<br />
[8] MERTENS, R. & WERMUTH, H. (1960) - Die<br />
Amphibien und Reptilien Europas. Dritte<br />
Liste,nach <strong>de</strong>m Stand vom 1. Januar 1960.<br />
Frankfurt am Main: Verlag Wal<strong>de</strong>mar Kramer.<br />
264.<br />
[9] COVACIU-MARCOV S.D, GHIRA I.,<br />
CICORT-LUCACIU A.D., SAS I.,<br />
STRUGARIU A., BOGDAN H.V. -<br />
Contributions to knowledge regarding the<br />
geographical distribution of the herpetofauna of<br />
Dobrudja, Romania. North-Western Journal of<br />
Zoology Vol. 2, No. 2, 2006, 88-125<br />
[10] FUHN, I.E. (1969) - Broaste, serpi, sopirle.<br />
Bucuresti: Ed. Natura si Omul. 246 [In<br />
Romanian].<br />
[11] COSSWHITE, D.L., S.F. FOX, and THILL<br />
R.E. 1999 - Comparison of methods for<br />
monitoring reptiles and amphibians in upland<br />
forests of the Ouachita mountains, Proceedings<br />
of the Oklahoma Aca<strong>de</strong>my of Science 79:45-50.<br />
118<br />
[12] CAMPBELL, H.W., and S.P. CHRISTMAN<br />
1982 - Field techniques for herpetofaunal<br />
community analysis. 193-200 in N. J. Scott, Jr.,<br />
ed. Herpetological Communities, U.S.D.I. Fish<br />
and Wildlife Service, Wildlife Research Report<br />
13, Washington, D.C. 239 .<br />
[13] RYAN, T.J., PHILIPPI, T., LEIDEN, Y.A.,<br />
DORCAS, M.E., WIGLEY, T.B. and<br />
[14] GASC, J.-P., CABELA, A., CRNOBRHJA-<br />
ISAILOVIC, J.,DOLMEN, D.,<br />
GROSSENBACHER, K., HAFFNER,<br />
P.,LESCURE, J., MARTENS, H., MARTINEZ-<br />
RICA, J.P., MAURIN, H., OLIVEIRA, M.E.,<br />
SOFIANIDOU, T.S., VEITH, M. &<br />
ZUIDERWIJK, A.(Eds.) 1997 - Atlas of<br />
Amphibians and Reptiles in Europe.<br />
[15] ENGE, K.M. 2001 - The pitfalls of pitfall traps.<br />
Journal of Herpetology 35(3): 467-478.<br />
[16] FERNER, J. W. 1979. A review of marking<br />
techniques for amphibians and reptiles, Society<br />
for the Study of Amphibians and Reptiles,<br />
Circular 9: 1-42.<br />
[17] KARNS, D.R. 1986 - Field herpetology:<br />
methods for the study of amphibians and<br />
reptiles, in Minnesota. James Ford Bell<br />
Museum of Natural History, occasional papers<br />
18.<br />
[18] CORN, P. S., and R. B. BURY. 1990 -<br />
Reptiles. USDA Forest Service, General and<br />
Technical Report PNW-GTR-256, 34.<br />
[19] ANDREI, M., 2002 - Contributions to the<br />
knowledge of the herpetofauna of southern<br />
Dobrudja (Romania). Trav. Mus. Nat. d'Hist.<br />
Nat. Gr. Antipa 44, 357-373<br />
[20] BOIAN, P.P. 2007 © Springer, Amphibians and<br />
Reptiles of Bulgaria: Fauna, Vertical<br />
Distribution and Consrvation, 85-107 , in<br />
Biogeography and Ecology of Bulgaria, V. Fet<br />
& A. Popov (eds.)
The present situation of the nose horned viper.../ Ovidius University Annals, Biology-Ecology Series 14: 115-120 (2010)<br />
Rusalka<br />
Yailata<br />
Kamen Bryag<br />
Kaliacra<br />
Bolata Dèrè<br />
Canaraua Fetii<br />
Hagieni<br />
Adamclisi<br />
Dumbrăveni<br />
Babadag<br />
Gura Dobrogei<br />
Măcin<br />
Priopcea<br />
Niculiţel<br />
Târguşor<br />
Cerna<br />
Albesti<br />
Şipotele<br />
Casimcea<br />
Atmagea<br />
Beştepe<br />
Cataloi<br />
0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00%<br />
119<br />
Juveniles<br />
Adults<br />
Fig. 2. The percentage of adults and juveniles in the analyzed populations
Marian Tudor / Ovidius University Annals - Biology-Ecology Series 14: 115-120 (2010)<br />
Canaraua Fetii<br />
Hagieni<br />
Adamclisi<br />
Dumbrăveni<br />
Babadag<br />
Gura Dobrogei<br />
Măcin<br />
Yailata<br />
Bolata Déré<br />
Kaliakra<br />
Priopcea<br />
Rusalka<br />
Kamen Bryag<br />
Niculiţel<br />
Târguşor<br />
Cerna<br />
Albesti<br />
Şipotele<br />
Casimcea<br />
Atmagea<br />
Beştepe<br />
Cataloi<br />
0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 16.00%<br />
Fig. 3. The abundance of Nose-Horned Viper in the analyzed populations<br />
120
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
BODY SIZE VARIATION IN RANA TEMPORARIA POPULATIONS<br />
INHABITING EXTREME ENVIRONMENTS<br />
Rodica PLĂIAŞU ** , Raluca BĂNCILĂ ** , Dan COGĂLNICEANU *<br />
* Ovidius University Constanţa, Faculty of Natural and Agricultural Sciences, Aleea Universităţii nr. 1,<br />
corp B, Constanţa 900470, Romania<br />
** “Emil Racoviţă” Institute of Speleology, 13 Septembrie Road, No. 13, Bucharest 050711, Romania<br />
___________________________________________________________________________<br />
Abstract: We studied the variation in body size in populations of a wi<strong>de</strong>spread anuran species, Rana<br />
temporaria, from high altitu<strong>de</strong> and latitu<strong>de</strong>s. Our results indicated a variable interannual pattern of body size,<br />
suggesting that body size in extreme environments is influenced by many factors. This indicates that long-term<br />
series of observations are nee<strong>de</strong>d to separate natural fluctuations from man-induced changes.<br />
Keywords: Rana temporaria, extreme environments, body size, interannual variation<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
During the last <strong>de</strong>ca<strong>de</strong>s, many amphibian<br />
species have <strong>de</strong>clined from high altitu<strong>de</strong> area, even in<br />
habitats apparently without human impact [1, 2]. The<br />
causes of some <strong>de</strong>clines remain unknown.<br />
Un<strong>de</strong>rstanding of the life history characteristics of<br />
the amphibian populations that inhabit extreme<br />
environments at high altitu<strong>de</strong> and latitu<strong>de</strong> is an<br />
important step in the evaluation process of the<br />
potential causes of <strong>de</strong>cline. Genetic and<br />
environmental factors (e.g. temperature, rainfall,<br />
trophic resources, competition, predators) <strong>de</strong>termine<br />
variation in the life history traits of species<br />
occupying a large geographic area [3]. Low<br />
temperature, associated with high altitu<strong>de</strong>/latitu<strong>de</strong>,<br />
reduces the activity period and the time available for<br />
resource exploitation [4]. Temperature affects the<br />
duration of hatching and metamorphosis in<br />
amphibians. The increase in the adult body size has<br />
been frequently associated with a cold annual<br />
temperature [5, 6]. Most studies of variation in<br />
amphibians body size have focused on latitudinal and<br />
altitudinal variation, e.g. trying to establish if the<br />
amphibian species follow the Bergmann’s rule [7, 8].<br />
Studies on the interannual variations in amphibians<br />
body size generally analyze difference in body<br />
condition [9, 10], or variation in age and size at<br />
maturity [6].<br />
The Common Frog (Rana temporaria) is the<br />
most wi<strong>de</strong>spread amphibian species in Europe [11].<br />
Its distribution reaches 71 o N in Fennoscandia [12]<br />
and it can be found even at altitu<strong>de</strong>s of 2600 m [12].<br />
The wi<strong>de</strong> altitudinal and latitudinal range of this<br />
species, allows comparisons of life-history traits over<br />
a broad range of conditions. In a previous publication<br />
we analyzed the altitudinal and latitudinal body size<br />
variation among populations from high altitu<strong>de</strong> and<br />
latitu<strong>de</strong> of R. temporaria testing if the variation<br />
pattern is according to the Bergmann’s rule [13]. In<br />
this study we analyzed interannual body size<br />
variation in the same Rana temporaria populations,<br />
in or<strong>de</strong>r to evaluate if the pattern of variation in body<br />
size changes in time. We tested the following<br />
predictions: i) there is no interannual variation in<br />
body size and ii) the mean body size of the frog<br />
populations from subarctic regions shows significant<br />
variation during a growth season.<br />
2. Material and Methods<br />
R. temporaria populations were studied from<br />
Kilpisjarvi, Finland (latitu<strong>de</strong> N 69 o ) in 2003 (August)<br />
and 2009 (July), Kolari, Finland (latitu<strong>de</strong> 67.2 o ) in<br />
2009 (July) and in Retezat National Park, Romania<br />
(latitu<strong>de</strong> N 45 o ) in 2004 (September) and 2009<br />
(August). Latitu<strong>de</strong> and altitu<strong>de</strong> were recor<strong>de</strong>d for<br />
each population by using a handheld Garmin GPS.<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010)<br />
Captured individuals were sexed, weighed (W)<br />
to the nearest 0.01 g with a portable electronic<br />
balance (AccuLab Pocket Pro), and snout-vent length<br />
(SVL) was measured to the nearest 0.5 mm with dialcalipers.<br />
Data were log transformed prior to analyses.<br />
For comparisons between years and sites we used<br />
One-way analysis of variance (ANOVA) and<br />
Analysis of covariance (ANCOVA) to compare the<br />
slopes of the regression lines.<br />
Statistical analyses were performed using SPSS ver.<br />
10.0 (SPSS Inc., 1999).<br />
3. Results and Discussions<br />
A total of 347 individuals were measured and<br />
weighed in 2003/2004, of which 237 juveniles and<br />
110 adults (67 m<strong>ale</strong>s and 43 fem<strong>ale</strong>s) and 157<br />
individuals in 2009 (66 juveniles, 43 fem<strong>ale</strong>s and 48<br />
m<strong>ale</strong>s). Both log transformed W and SVL were<br />
normally distributed (W: D = 5.06, p < 0.001;<br />
122<br />
SVL: D = 3.75, p < 0.001). The body size indices of<br />
the studied populations are presented in Tables 1 and<br />
2. There was no significant difference in SVL<br />
between Retezat National Park and Finland -<br />
Kilisjarvi populations. We found significant<br />
differences in the mean body size indices between<br />
the two stations from Finland (Table 3).<br />
We then compared W and SVL from different years<br />
for the same population. We found significant<br />
differences in the interannual variation of the body<br />
size indices for juveniles in both Finland and Retezat<br />
populations, and in the mean weight for fem<strong>ale</strong>s.<br />
M<strong>ale</strong>s showed only in Retezat a significant<br />
interannual variation in the body size indices (Table<br />
4). We also compared the slopes of the regression<br />
lines of W as a function of SVL. The slopes of the<br />
regression lines are significantly different for all<br />
adults in Retezat and Finland (Fig. 1: F1,56= 81.41,<br />
p
Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010)<br />
Table 3. Comparison of SVL and W between the three R. temporaria populations, by using ANCOVA (FN =<br />
Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitu<strong>de</strong>; N = sample size, *P < 0.05, ***P <<br />
0.001, NS = not significant).<br />
W SVL<br />
N Average Fa Average Fa<br />
FN vs FS FN FS FS FN FS FN<br />
Fem<strong>ale</strong> 13 16 18.956 14.308 6.069* 71.244 61.154 26.384***<br />
M<strong>ale</strong> 17 9 17.267 15.853 0.737 NS<br />
69.89 62.46 22.88***<br />
Juveniles 50 9 4.333 7.779 10.381* 42.17 45.95 1.748 NS<br />
FN vs RNP FN RNP FN RNP FN RNP<br />
Fem<strong>ale</strong> 13 14 14.308 34.69 19.504*** 61.154 76.78 3.627 NS<br />
M<strong>ale</strong> 17 22 15.853 36.20 17.93*** 62.46 73.41 1.778 NS<br />
Juveniles 50 7 7.779 9.28 5.903* 45.95 44.8 0.198 NS<br />
Table 4. Comparison of the interannual variation in SVL and W between the three R. temporaria populations, by<br />
using ANCOVA (FN = Finland - Kilpisjarvi; FS = Finland - Kolari, RNP = Retezat high altitu<strong>de</strong>; N = sample<br />
size, *P < 0.05, ***P < 0.001, NS = not significant).<br />
W SVL<br />
N Average Fa Average Fa<br />
FN 2003 vs. 2009 2003 2009 2003 2009 2003 2009<br />
Fem<strong>ale</strong> 29 13 31 14.308 4.235* 65.94 61.154 3.70 NS<br />
M<strong>ale</strong> 32 17 31.76 15.853 3.713 NS<br />
67.59 62.46 0.43 NS<br />
Juveniles 197 50 3.17 7.779 78.41*** 29.01 45.95 120.01***<br />
RNP 2004 vs. 2009 2004 2009 2004 2009 2004 2009<br />
Fem<strong>ale</strong> 14 14 61.16 34.69 14.56*** 82.7 76.78 2.24 NS<br />
M<strong>ale</strong> 35 22 47.14 36.20 12.21*** 77.07 73.41 4.49*<br />
Juveniles 40 7 2.19 9.28 22.56*** 24.42 44.8 19.4***<br />
The variation in the adult body size reported in<br />
amphibians can be induced by several factors,<br />
including genetic and environmental differences,<br />
such as: duration of the activity period, food<br />
availability and climatic conditions [6, 14]. Laugen<br />
et al. (2005) found that body size <strong>de</strong>creased with<br />
latitu<strong>de</strong> in the Scandinavian Common Frog<br />
populations. Comparisons between populations from<br />
Western Europe with different activity periods report<br />
increases in the mean length, as activity period gets<br />
shorter [6]. Rana temporaria populations from the<br />
analyzed area show a variable pattern in weight and<br />
length. Băncilă et al. (2010) found that latitudinal and<br />
altitudinal variation patterns in juvenile body size<br />
123<br />
were according to the Bergmann’s rule. We found an<br />
opposite pattern for juveniles, with <strong>de</strong>creases in the<br />
body size as the activity period gets shorter. Since<br />
juveniles have higher growth and <strong>de</strong>velopment rate<br />
than adults, difference could be observed even in the<br />
case of short periods of time between sampling.<br />
Interpopulational variation in the adult body size<br />
could be caused by differences in the age structure.<br />
Growth rates in amphibian species can dramatically<br />
<strong>de</strong>crease after the attainment of sexual maturity (e.g.<br />
Miaud et al. 1999). Thus, <strong>de</strong>layed reproduction can<br />
allow a prolonged growth period and the attainment<br />
of a larger adult size.
Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010)<br />
W (log)<br />
2.0<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
1.75 1.80 1.85 1.90 1.95 2.00<br />
SVL (log)<br />
RNP 2004<br />
RNP 2009<br />
Fig. 1. Body size indices for m<strong>ale</strong>s in RNP<br />
populations, 2004 (N=35; R 2 = 0.72) and 2009 (N=22;<br />
R 2 = 0.90).<br />
W (log)<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
1.65 1.70 1.75 1.80 1.85 1.90 1.95<br />
SVL (log)<br />
Kilpisjarvi 2003<br />
Kilpisjarvi 2009<br />
Fig. 3. Body size indices for fem<strong>ale</strong>s in Finland-<br />
Kilpisjarvi, 2003 (N=29; R 2 = 0.63) and 2009 (N=13;<br />
R 2 = 0.59).<br />
W (log)<br />
1.6<br />
1.5<br />
1.4<br />
1.3<br />
1.2<br />
1.1<br />
1.0<br />
0.9<br />
Kolari<br />
Kilpisjarvi<br />
1.70 1.75 1.80 1.85 1.90 1.95<br />
SVL (log)<br />
Fig. 5. Body size indices for fem<strong>ale</strong>s in Finland<br />
Kilspijarvi (N=13; R 2 = 0.59) and Finland Kolari 2009<br />
(N=16; R 2 = 0.71).<br />
124<br />
W (log)<br />
2.2<br />
2.0<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
1.70 1.75 1.80 1.85 1.90 1.95 2.00 2.05<br />
SVL (log)<br />
RNP 2004<br />
RNP 2009<br />
Fig. 2. Body size indices for fem<strong>ale</strong>s in RNP<br />
populations, 2004 (N=14; R 2 = 0.75) and 2009 (N=14;<br />
R 2 = 0.95).<br />
W (log)<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
Kilpisjarvi 2003<br />
Kilpisjarvi 2009<br />
1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88<br />
SVL (log)<br />
Fig. 4. Body size indices for m<strong>ale</strong>s in Finland-<br />
Kilpisjarvi, 2003 (N=32; R 2 = 0.70) and 2009 (N=17;<br />
R 2 = 0.63).<br />
W (log)<br />
1.5<br />
1.4<br />
1.3<br />
1.2<br />
1.1<br />
1.0<br />
0.9<br />
Kolari<br />
Kilpisjarvi<br />
1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90<br />
SVL (log)<br />
Fig. 6. Body size indices for m<strong>ale</strong>s in Finland<br />
Kilpisjarvi (N=17; R 2 = 0.89) and Finland Kolari 2009<br />
(N=9; R 2 = 0.70).
Rodica Plăiaşu et al. / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010)<br />
Factors such as temperature and humidity can<br />
directly affect the activity period and the<br />
availability of food, influencing the growth rate and<br />
the fat stores; hence they could consequently<br />
<strong>de</strong>termine significant interannual variation in the<br />
body size. Populations from both analyzed areas<br />
exhibit interannual variation in weight and length.<br />
This variation mainly affects the weight and could<br />
be the result of the differences in the sampling<br />
period. The pattern of the adult size variation could<br />
also directly result from the variation in the<br />
population age structure. Further analyses are<br />
necessary to <strong>de</strong>termine whether variation in the age<br />
structure are contributing or not to the interannual<br />
body size indices. Results suggest that many factors<br />
affect the body size in extreme environment and<br />
long-term series of observations are nee<strong>de</strong>d in or<strong>de</strong>r<br />
to separate natural fluctuations from the human<br />
impact/global warming.<br />
4. Conclusions<br />
This study stresses the importance of analyzing<br />
interannual variation of life history traits, because<br />
one-year data may not properly reflect the features<br />
of a population and this issue becomes important in<br />
the context of global changes and their possible<br />
effects on the amphibian populations.<br />
Acknowledgements<br />
The research was fun<strong>de</strong>d by the EU FP6<br />
(Lapbiat) and EU FP7 (Lapbiat 2) Romanian<br />
CNCSIS grant 1114/2004. We are grateful to<br />
Claudia Jianu, Dorel Ruşti, Ioan Ghira and Marian<br />
Tudor for their help during fieldwork.<br />
5. References<br />
[1] LAURANCE W.F., McDonald K.R., Speare R.,<br />
1996 - Epi<strong>de</strong>mic disease and the catastrophic<br />
<strong>de</strong>cline of Australian rain forest frogs.<br />
Conservation Biology, 10: 406-413.<br />
[2] YOUNG B.E., Lips K.R., Reaser J.K., Ibanez<br />
R., Salas A.W., Ce<strong>de</strong>no J.R., Coloma L.A., Ron<br />
S., La Marca E., Meyer J.R., Munoz A., Bolanos<br />
F., Chaves G., Romo D. 2001. Population<br />
<strong>de</strong>clines and priorities for amphibian<br />
125<br />
conservation in Latin America. Conservation<br />
Biology, 15: 1213-1223.<br />
[3] SORCI G., Clobert J., Belichon S., 1996 -<br />
Phenotypic plasticity of growth and survival in<br />
the common lizard Lacerta vivipara. Journal of<br />
Animal Ecology, 65: 781-790.<br />
[4] RYSER J., 1996 - Comparative life histories of<br />
a low- and a high-elevation population of the<br />
common frog Rana temporaria. Amphibia–<br />
Reptilia, 17: 183-195.<br />
[5] FICETOLA G.F., Scali S., Denoël M.,<br />
Montinaro G., Vukov T.J., Zuffi M.A.L., Padoa-<br />
Schioppa E., 2010 - Ecogeographical variation of<br />
body size in the newt Triturus carnifex:<br />
comparing the hypotheses using an informationtheoretic<br />
approach. Global Ecology and<br />
Biogeography, 19: 485-495.<br />
[6] MIAUD C., Guyétant R., Elmberg J., 1999 -<br />
Variations in life-history traits in the common<br />
frog (Rana temporaria) (Amphibia: Anura): a<br />
literature review and new data from the French<br />
Alps. Journal of Zoology, 249: 61-73.<br />
[7] ADAMS D.C., Church J.O., 2008 - Amphibians<br />
do not follow Bergmann’s rule. Evolution, 62:<br />
413-420.<br />
[8] ASHTON K.G., 2002 - Do amphibians follow<br />
Bergmann’s rule? Canadian Journal of Zoology,<br />
80: 708-716.<br />
[9] TOMAŠEVIĆ N., Cvetković D., Aleksić I.,<br />
Crnobrnja-Isailović J., 2007 - The effect of<br />
climatic conditions on post-hibernation body<br />
condition and reproductive traits of Bufo bufo<br />
fem<strong>ale</strong>s. Archives of Biological Sciences,<br />
Belgra<strong>de</strong>, 59: 51-52.<br />
[10] TOMAŠEVIĆ N., Cvetković D., Miaud C.,<br />
Aleksić I., Crnobrnja-Isailović J., 2008 -<br />
Interannual variation in life history traits between<br />
neighbouring populations of the wi<strong>de</strong>spread<br />
amphibian Bufo bufo. Revue d’Ecologie (Terre et<br />
Vie), 63: 371-381.<br />
[11] GASC J.P., Cabela A., Crnobrnja-Isailovic J.,<br />
Dolmen D., Grossenbacher K., Haffner P.,<br />
Lescure J., Martens H., Martínez Rica J.P.,<br />
Maurin H., Oliveira M.E., Sofianidou T.S., Veith<br />
M., Zui<strong>de</strong>rwijk A. (ed), 1997 - Atlas of<br />
Amphibians and Reptiles in Europe. Collection<br />
Patrimoines Naturels, 29, Societas Europaea<br />
Herpetologica, Muséum National d'Histoire
Body size variation in Rana tempoaria populations / Ovidius University Annals, Biology-Ecology Series 14: 121-126 (2010)<br />
Naturelle & Service du Petrimone Naturel, Paris,<br />
496 pp.<br />
[12] MERILÄ J., Laurila A., Laugen A.T.,<br />
Rasanen K., Pahkla M., 2000 - Plasticity in age<br />
and size at metamorphosis in Rana temporaria -<br />
comparison of high and low latitu<strong>de</strong> populations.<br />
Ecography, 23: 457-465.<br />
[13] BĂNCILĂ R.I., Plăiaşu R., Cogălniceanu, D.,<br />
2010 - Effect of latitu<strong>de</strong> and altitu<strong>de</strong> on body size<br />
in the common frog (Rana temporaria)<br />
126<br />
populations. Studii <strong>şi</strong> Cercetări, Biologie,<br />
Universitatea din Bacău,17: 43-46.<br />
[14] MORRISON C., Hero J., 2002 - Geographic<br />
variation in life history characteristics of<br />
amphibians: a review. Journal of Animal<br />
Ecology, 72: 270-279.<br />
[15] LAUGEN A.T., Laurila A., Jönsson K.I.,<br />
Sö<strong>de</strong>rman F., Merilä J., 2005 - Do common frogs<br />
(Rana temporaria) follow Bergmann’s rule?<br />
Evolutionary Ecology Research, 7: 717-731.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
UTILIZATION OF EPIFLUORESCENCE MICROSCOPY AND DIGITAL IMAGE<br />
ANALYSIS TO STUDY SOME MORPHOLOGICAL AND FUNCTIONAL ASPECTS<br />
OF PROKARYOTES<br />
Simona GHIŢĂ ** , Iris SARCHIZIAN * , Ioan ARDELEAN ***<br />
* Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology,<br />
Mamaia Avenue, No. 124, Constanţa, 900552, Romania,<br />
e-mail: ghitasimona@aim.com, irissarchizian@yahoo.com<br />
** Constanta Maritime University, Department of Environmental Engineering, Mircea cel Batrin, No. 104,<br />
Constanta, 900663, Romania, e-mail:ghitasimona@aim.com;<br />
*** Institute of Biology, Splaiul In<strong>de</strong>pen<strong>de</strong>nţei, No. 296, Bucharest, 060031, Romania,<br />
email:ioan.ar<strong>de</strong>lean57@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: The aims of this study is to argue, based on original results, the importance of utilization of<br />
epifluorescence microscopy to study some morphological and functional aspects of prokaryotes allowing to<br />
perform total cell counts , direct viable count count, count of permeabilised cells, chlorophyll containing cells or<br />
putatively capsulated cells. Automated image analysis of the results thus obtained was done using CellC and<br />
ImageJ software which allow the quantification of bacterial cells from digital microscope images, automated<br />
enumeration of bacterial cells, comparison of total count and specific count images, providing also quantitative<br />
estimates of cell morphology.<br />
Keywords: epifluorescence, digital image analysis, heterotrophic bacteria, cyanobacteria.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The use of epifluorescence microscopy to study<br />
different aspects of prokaryotes at population and<br />
single cell level significantly improved the<br />
knowledge concerning which species are present in a<br />
given sample, the cell <strong>de</strong>nsity and the metabolic<br />
statues of the population as a whole or of each single<br />
prokaryote cell (Van Wambeke, 1995; Manini &<br />
Danovaro, 2006; Falcioni et al., 2008; Kirchman,<br />
2008; Ar<strong>de</strong>lean et al., 2009). In the last <strong>de</strong>ca<strong>de</strong>s there<br />
is also an increase in the <strong>de</strong>velopment and use of<br />
different softwares for automated analysis of the<br />
digital images thus obtained (Ishii et al. 1987; Estep<br />
& Macintyre 1989; Embleton et al., 2003; Walsby,<br />
1996; Congestri et al. 2003; Selinummi et al., 2005,<br />
2008).<br />
The aims of this study is to argue, based on<br />
original results, the importance of utilization of<br />
epifluorescence microscopy coupled with automated<br />
image analysis to study some morphological and<br />
functional aspects of prokaryotes allowing to<br />
perform total cell counts (acridine orange, DAPI,<br />
SYBR Green 1), direct viable count (elongated cell in<br />
the presence of nalidixic acid, labelled with acridine<br />
orange), count of permeabilised cells (cells<br />
permeable to propidium iodi<strong>de</strong>), putatively<br />
capsulated cell (labelled with aniline blue) and<br />
chlorophyll containing cells both in enriched cultures<br />
and in natural (microcosms) samples.<br />
2. Material and Methods<br />
A. Study area and sampling. Samples were<br />
collected in sterile bottles in October 2008 and May<br />
2009 from sulphurous mesothermal spring (Obanul<br />
Mare) placed in north of Mangalia City<br />
(43˚49’53.6’’N; 28˚34’05.3’’E). The samples were<br />
divi<strong>de</strong>d in sub-samples, one being immediately fixed<br />
with buffered formal<strong>de</strong>hy<strong>de</strong> (2% final concentration)<br />
and the second one used to isolate cyanobacteria by<br />
inoculation into conical flasks with either BG11<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
medium or nitrate - free BG11 medium (BG0)<br />
(Rippka et al., 1979). Another series of natural<br />
samples were collected from Black Sea (Tomis<br />
seaport at 0.5m <strong>de</strong>pth; 44 o 10 ’ 44 ’’ N; 28 o 39 ’ 32 ’’ E) in<br />
March 2009.<br />
B. Culture conditions. Natural samples<br />
inoculated in either BG11 or BG0 media, either solid<br />
of liquid, were incubated in culture room at 25 ± 1ºC<br />
and illuminated with fluorescent tubes having the<br />
photon rate of 50 μmol m –2 s –1 at surface of the<br />
culture vessels.<br />
C. Microcosms. Taking into account the<br />
advantages of microcosms (Iturbe et al., 2003;<br />
Molina-Barahona et al., 2004) we used this<br />
opportunity as previously (Ar<strong>de</strong>lean et al., 2009).<br />
D. Total cell count (AO; DAPI, SYBR Green<br />
I)<br />
Total bacterial count were performed using<br />
acridine orange, DAPI and SYBR Green I (Luna et<br />
al., 2002; Lunau et al., 2005; Manini & Danovaro,<br />
2006).<br />
For AO and DAPI (5 μg/mL dye final<br />
concentrations) subsamples were stained for 5<br />
minutes and were filtred on black Millipore 0,22µm<br />
pore size filters. Unlike AO, using DAPI for bacterial<br />
visualization and enumeration has the advantages of<br />
low background fluorescence and that DAPI stains<br />
only DNA.<br />
For SG (1µL/10µL sample final concentrations)<br />
subsamples were stained for 10 minutes and were<br />
filtred on black Millipore 0,22 µm pore size filters.<br />
Color filters were washed with 10 ml of 17 ‰ saline<br />
solution. SG as a permeant DNA-binding stain and<br />
<strong>de</strong>termine the total fraction of cells from natural<br />
samples.<br />
E. Permeabilized (<strong>de</strong>ad) cells (PI+)<br />
PI is a double-charged phenanthridium<br />
<strong>de</strong>rivative and is one of the most common stains for<br />
<strong>de</strong>ad cells (Luna et al., 2002). PI is thus assumed to<br />
be unable to penetrate cell membranes. In our natural<br />
samples we used a PI concentration of 5 μL/ml<br />
sample. Also stained samples were filtered through<br />
black Millipore 0,22 µm pore size filters and then<br />
inspected un<strong>de</strong>r a epifluorescence microscope. The<br />
disruption of planktonic cell aggregates for cell<br />
enumeration were done as previously shown<br />
(Ar<strong>de</strong>lean et al., 2009).<br />
128<br />
F. Enumeration of (putatively) capsulated<br />
cells (AB+). Cell capsule was also inspected using<br />
aniline blue (AB) which is a fluorescent dye specific<br />
which seems to be specific for 1,3 beta glucans<br />
(Hong et al., 2001) found in plants and as capsular<br />
material in many microorganisms (Nakanishi et al.,<br />
1976; McIntosh et al., 2005). Capsular envelopes are<br />
wi<strong>de</strong>ly distributed in marine free-living and particleassociated<br />
bacteria (Heissenberger et al., 1996) and<br />
are a signature of active bacteria (Sto<strong>de</strong>regger &<br />
Herndl, 2002). Bacteria with an intact intracellular<br />
structure, and therefore potentially active bacteria,<br />
are surroun<strong>de</strong>d by a capsular layer, while the vast<br />
majority of bacteria with a damaged structure lack<br />
such a capsule (Heissenberger et al., 1996).<br />
Laboratory experiments indicated that active bacteria<br />
are constantly renewing their capsular envelope and<br />
releasing a significant fraction of the polysacchari<strong>de</strong><br />
layer into the ambient water (Sto<strong>de</strong>regger & Herndl,<br />
2002). The samples were treated with AB (5 µg/mL<br />
final concentration) for 5 minutes and then filtered<br />
and counted as shown above for AO staining .<br />
G. The automatic cell analysis were done with<br />
two software ImageJ and CellC, who was applied to<br />
digital images of whole cells color-stained bacteria<br />
and cyanobacteria. The analysis proceeds few<br />
important steps: the background is separated from the<br />
objects based on the intra-class variance threshold<br />
method; noise and specks of staining color in the<br />
image can affect the reliability of the analysis, so<br />
those was removed. The removal was done applying<br />
mathematical morphology operations to the image;<br />
then separation of clustered objects was performed<br />
(Selinummi, 2008). The length of cells was<br />
<strong>de</strong>termined with ImageJ software using a calibration<br />
sc<strong>ale</strong>.<br />
H. Cyanobacteria (natural fluorescence)<br />
Visualization of hydrocarbon tolerant<br />
phototrophic microorganisms, also for unicellular or<br />
filamentous cyanobacteria from sulphurous<br />
mesothermal spring; chlorophyll a in natural<br />
environments (either marine or spring) was done<br />
using an epifluorescence microscope (N-400FL, lamp<br />
Hg 100W, type on the blue filter; Sherr et al., 2001)<br />
as previously shown (Ar<strong>de</strong>lean et al., 2009).<br />
I. Direct viable count (cells capable of<br />
division) is based on the Kogure method <strong>de</strong>velopped
Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
by incubation of samples with a single antimicrobial<br />
agent (nalidixic acid) and nutrients (yeast extract).<br />
Nalidixic acid acts as a specific inhibitor of DNA<br />
synthesis and prevents cell division without affecting<br />
other cellular metabolic activities, including cell<br />
growth; thus viable cells growth but do not divi<strong>de</strong>,<br />
thus becoming longer/larger than cells unable to grow<br />
(Kogure et al., 1979). Experiments were done with<br />
40mL samples from each microcosms in which the<br />
sample was filtered through 0.45 µm filter (2 and 3)<br />
supplemented with yeast extract (50 mg / L final<br />
concentration), nalidixic acid (20 mg / L final<br />
concentration) (Kogure et al., 1979) and gasoline<br />
(0.5% final concentration); 17 hours before the start<br />
of experiment all samples were kept in an incubator<br />
at a temperature of 30 o C and continuous stirring.<br />
Subsequently samples were incubated un<strong>de</strong>r the<br />
conditions previously reported and samples were<br />
harvested each two hours (consi<strong>de</strong>ring the time To,<br />
T1 –after 2 hours, T2- after 4 hours; T3- after 6 hours,<br />
T4- after 8 hours).<br />
3. Results and Discussions<br />
1. Total count cell (AO+, DAPI+), permeable<br />
(<strong>de</strong>ad) cells (PI+) and (putative) capsulated cells<br />
(AB+)<br />
In experimental microcosms we viewed the<br />
gasoline tolerant/oxidizing bacteria to make a clear<br />
distinction between the total number of cells (stained<br />
with AO, DAPI), the number of encapsulated, active<br />
cells, (AB +), and the number of permeable (PI+) ,<br />
<strong>de</strong>ad (figures 1 and 3).<br />
Fig 1. Comparison between the total cell count (AO+<br />
and DAPI+) and permeable cells <strong>de</strong>nsity (IP+)<br />
129<br />
As shown in figure 1 the total number of<br />
heterotrophic cells counted using AO or DAPI is<br />
practically the same. Quantification was performed<br />
on samples previously fixed in experimental<br />
microcosms (M1 and M2). Comparing the total<br />
number of bacterial cells obtained with AO and<br />
DAPI stained (20 μL/mL sample) in experimental<br />
microcosms, we have shown that there are no<br />
significant differences in the use of two<br />
fluorochromes on natural samples (M1: 13.719,5<br />
cells ml -1 – SD (±39,7) AO and 13.494,6 cells ml -1 -<br />
SD (±22,2) DAPI, respectively M2: 14.619,1 cells<br />
ml -1 – SD (±28,8) AO and 15.787,4 cells ml -1 SD<br />
(±32,8) DAPI).<br />
Fig 2. Total number of cells obtained using AO and<br />
SG in natural sample<br />
To assess the number of cells obtained after<br />
staining AO and SG, we used unfixed samples<br />
collected from microcosm 1.<br />
As shown in figure 2 there are differences in<br />
total counts obtained by the use of either AO or<br />
SYBR Green 1, the higher count obtained with the<br />
last fluorochrome (47,6%) being due to its higher<br />
fluorescence yield, in agreement with international<br />
literature (Weinbauer et al., 1998; Luna et al., 2002;<br />
Lunau et al., 2005 ), allowing the visualization of<br />
smaller cells.<br />
As shown in figure 1, the number of <strong>de</strong>ad cells<br />
(PI positive) is 12.3% of the total number obtained<br />
with the two fluorochromes, AO and DAPI.<br />
In figure 3 one can see the cell <strong>de</strong>nsities of<br />
putative capsulated cells which are 10% from the<br />
<strong>de</strong>nsity obtained with acridine orange.
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
Fig 3. Aniline blue positive cells as compared with<br />
acridine orange positive cells.<br />
2. Cyanobacteria (natural fluorescence)<br />
Natural fluorescence of these prokaryotic in<br />
various natural environments (marine and sulphurous<br />
mesothermal spring) and marine microcosms was<br />
studied by epifluorescence microscopy (figure 4).<br />
a b<br />
c d<br />
Fig 4. Natural fluorescence of gasoline-tolerant<br />
oxygenic phototrophic microorganism from<br />
microcosm 2 supplemented with gasoline (a);<br />
microcosm 1 supplemented with gasoline and<br />
nutrient (b) and microorganism isolated from<br />
sulphurous mesothermal spring Obanul Mare<br />
(Mangalia) (c and d).<br />
In microcosm 1 cyanobacteria filaments are<br />
much thinner (1.35 ± 0.27) compared with<br />
microcosm 2 (3.87 ± 0.57) (fig.5); the significance of<br />
this difference being un<strong>de</strong>r investigation.<br />
130<br />
Fig 5. Filaments of cyanobacteria in the M2.<br />
3. Direct viable count<br />
In figure 6 there are presented the results<br />
concerning changes in average cell lengths of<br />
bacterial populations from the two microcosms with<br />
filtered water (0,45µm) each supplemented with yest<br />
extract, nalidixic acid and gasoline (see Materials and<br />
methods).<br />
Fig 6. Average length of cells from To to T4 (after 8<br />
hours of incubation with nalidixic acid) in the two<br />
microcosms (M2 and control, M3)<br />
As can be seen in Figure 6, after 8 hours of<br />
incubations, the average length of M2 cells is about 7<br />
µm, compared with the M3 where the cells were<br />
maintained in high proportion in the form of cocci<br />
(average diameter of about 2 µm). These results<br />
argue the possibility to count viable cells, cells able<br />
to grow, by a relatively simple method. It seems<br />
appropriate to assume that the large increase in cell<br />
size in bacterial populations which have been<br />
previously selected to grow in the presence of<br />
gasoline (microcosms 2) is due to the cells ability to<br />
oxidize/tolerate gasoline, as compared with the<br />
populations sampled from the control microcosms<br />
where the proportion of gasoline tolerant bacteria is
Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
very low (and responsible for the low increase in the<br />
average cell lengths in M3). In M3 (control) one can<br />
see a rather constant length of some cells during<br />
incubation (2,15±0,37) whereas in M2 there was a<br />
sud<strong>de</strong>n increase in cell length (6,46±1,54) to the time<br />
T2 (4 hours incubation) then there was a steady<br />
increase until T4 (8 hours incubation).<br />
In Figure 7 are some random fields of cells in<br />
the two microcosms to highlight how cell elongation<br />
occurred from the T o to T4 only in M2.<br />
a b<br />
c d<br />
Fig 7. Evaluating cell elongation in microcosm 2 (a-<br />
To and b-T4) respectively in the control microcosm<br />
(c- To and d-T4)<br />
Digital Image Analysis and automated image<br />
analysis for epifluorescence<br />
The automated approach will not only remove<br />
the need for tedious manual analysis work, but also<br />
enable biologists to measure cellular features not<br />
feasible by the standard manual techniques<br />
(Selinummi, 2008).<br />
In our studies we used ImageJ software - a<br />
public domain Java image processing and analysis<br />
program inspired by NIH Image for the Macintosh,<br />
who runs, either as an online applet or as a<br />
downloadable application, on any computer with a<br />
Java 1.5 or later virtual machine. This software was<br />
used to display, edit, analyze, process, save and print<br />
8–bit, 16–bit and 32–bit epifluorescence digital<br />
images, many image formats including TIFF, GIF,<br />
JPEG, BMP, supporting ‘stacks’and hyperstacks, a<br />
series of images that share a single window.<br />
131<br />
For study bacteria and cyanobacteria from our<br />
samples ImageJ was the main software for measure<br />
the length of cells and pixel value statistics of user<strong>de</strong>fined<br />
selections, creating <strong>de</strong>nsity histograms and<br />
line profile plots, supports standard image processing<br />
functions such as contrast manipulation, sharpening,<br />
smoothing, edge <strong>de</strong>tection and median filtering.<br />
Digital images are two-dimensional grids of<br />
pixel intensities values with the width and height of<br />
the image being <strong>de</strong>fined by the number of pixels in x<br />
(rows) and y (columns) direction. Thus, pixels<br />
(picture elements) are the smallest single components<br />
of images, holding numeric values – pixel intensities<br />
– that range between black and white (ImageJ user<br />
gui<strong>de</strong>). Microphotographs used in this study was<br />
RGB images, RGB/HSB stacks, and composite<br />
images.<br />
People can see color with significant variations<br />
and the popular phrase “One picture is worth ten<br />
thousand words” may not apply to certain color<br />
images, especially those that do not follow the basic<br />
principles of Color Universal Design. That why this<br />
combining digital image analysis and automated<br />
analysis methods was usefull to distinguish some<br />
morphological and functional aspects of prokaryotes.<br />
We displied with ImageJ simultaneously several<br />
selections or regions of interest named ROIs, who can<br />
be measured, drawn or filled. Selections was initially<br />
outlined in one of the nine ImageJ <strong>de</strong>fault colors<br />
(Red, Green, Blue, Magenta, Cyan, Yellow, Orange,<br />
Black and White) and then, once created, selections<br />
was contoured or painted with any other color. Most<br />
of ImageJ analyses was printed to the Results table.<br />
Fig 8. The ImageJ Window<br />
(http://rsbweb.nih.gov/ij/).<br />
Straight Line Selection with “Alt” from<br />
computer keeps the line length fixed while moving<br />
either end of the line and forces the two points that<br />
<strong>de</strong>fine the line to have integer coordinate values when<br />
creating a line on a zoomed image.<br />
The CellC software is the second software used<br />
in automated analysis of our microscopy images like<br />
cell enumeration and measurements of cell’s<br />
properties (size, shape, intensity). We applied the
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
algorithms of CellC software for digital images,<br />
because this have three important parts: a MATLAB<br />
figure file of the segmented image (this can be<br />
exported in any common image file format; a comma<br />
separated value (CSV) - file with quantitative data of<br />
the cells (was opened in a spreadsheet program Excel<br />
for further analysis); a summary CSV-file with the<br />
cell count for each of the analyzed images for a quick<br />
overview of the analysis process (this file were only<br />
saved in the batch processing mo<strong>de</strong>). Fluorescence<br />
microscopy digital images were analyzed and the<br />
objects has different intensity than the background.<br />
Commonly, this property holds true for images of<br />
bacteria (http://sites.google.com/site/cellcsoftware/).<br />
Fig 9. CellC’s interface<br />
(http://sites.google.com/site/cellcsoftware/) used for<br />
automated digital analysis of bacteria/cyanobacteria.<br />
Furthermore, CellC software were used for two<br />
important purposes: to calculate total object count<br />
(e.g. DAPI stained cells) and co-localization analysis,<br />
comparing total and specific count images of the<br />
same location. When two images were analyzed, the<br />
co-localization was measured by comparing which<br />
cells are present only in the first image, and which are<br />
visible in both of the images. The binarized result<br />
images was saved as JPG-images, and the<br />
enumeration results and statistics are saved as an<br />
Excel-ready CSV file. The images was processed<br />
one at a time, or automatically in a batch.<br />
Graphical illustration of the analysis process<br />
and a part of a CSV-file opened in a spreadsheet<br />
program are given in Figure 9. The CSV-file gives,<br />
for each cell in the image, size and intensity<br />
information as well as information on cell<br />
morphologies. All results produced with digital image<br />
processing algorithms are perfectly reproducible.<br />
The image processing methods used guarantee<br />
that all images are analyzed using the same criteria,<br />
and therefore results between different images are<br />
comparable. CellC software is easy to use due to the<br />
132<br />
inclu<strong>de</strong>d graphical user interface, and the batch<br />
processing mo<strong>de</strong> enables fast and convenient<br />
processing of hundreds of cell images.<br />
CellC enumerate bright cells on a dark<br />
background (epifluorescence). We also used two<br />
different methods to process the images: one<br />
image/image pair at a time; several images pairs<br />
sequentially in batch processing mo<strong>de</strong>.<br />
If the background of the image is uneven (because of<br />
e.g. misaligned lighting), it is preferable to choose<br />
this option.<br />
The <strong>de</strong>fault option in CellC is to present the<br />
measured parameters in pixels. By checking this box<br />
we <strong>de</strong>fine how many micrometers one pixel<br />
corresponds to, and receive all measurement results<br />
in micrometers. The correct value of this setting<br />
obviously <strong>de</strong>pends on the imaging setup, such as on<br />
the camera and the objective, and must be <strong>de</strong>termined<br />
outsi<strong>de</strong> CellC, using ImageJ to calibrate the sc<strong>ale</strong>.<br />
The main technical requirement for using CellC<br />
is the clear visual distinction between the cells to be<br />
counted and their background, which could be<br />
achieved relatively easy by epifluorescence<br />
microscopy (Ar<strong>de</strong>lean et al., 2009).<br />
If darker regions exist insi<strong>de</strong> cells, thresholding<br />
may result in false holes insi<strong>de</strong> cells (darker pixels<br />
are consi<strong>de</strong>red background). By selecting this option,<br />
these holes are automatically filled. Sometimes the<br />
fill can cause worse cell cluster separation results.<br />
Automatic removal of over/un<strong>de</strong>rsized cells<br />
were selected, because CellC automatically <strong>de</strong>ci<strong>de</strong>s<br />
which particles are too small to be consi<strong>de</strong>red as real<br />
cells. All <strong>de</strong>tected objects that are smaller than 1/10<br />
of the mean size of all objects, were removed.<br />
Because the sizes of un<strong>de</strong>r/oversized particles were<br />
known using “Analyze Measure” option of ImageJ, it<br />
was possible to set the thresholds manually by using<br />
the text boxes. The unit of sizes <strong>de</strong>pends on the user<br />
<strong>de</strong>fined unit (pixels/μm 2 ).<br />
The CSV data sheet consists of following<br />
columns: cell's serial number (a unique number given<br />
to each cell); area of cell (estimate of the cell area);<br />
approximate volume (approximation of the volume of<br />
the cell); length (estimate of the cell length); width<br />
(estimate of the cell width); intensity mean, (mean<br />
intensity of the cell); intensity maximum, (maximum<br />
intensity of the cell); solidity (estimate of the shape of<br />
the cell); compactness (estimate of the shape of the
Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
cell). Means of each column and the unit of measure<br />
(pixels or micrometers) are presented in the end of<br />
the file.<br />
Image acquisition—Images for analysis were<br />
done with a Canon digital camera. Brightness and<br />
contrast were adjusted for the first image and kept<br />
unchanged throughout the image acquisition<br />
procedure. The images (1600 by 1200 pixels, 256<br />
dpi) were acquired at 50x magnification and stored as<br />
543-KB JPG files. Additional images acquired at<br />
100x magnification were used to verify that<br />
measurements of individual filaments/ bacteria were<br />
in<strong>de</strong>pen<strong>de</strong>nt of magnification<br />
a) Acridin-orange stained filamentous<br />
cyanobacteria isolated from mesothermal sulfurous<br />
spring were analysed using Image J software for<br />
distinguish heterocystous cells. First of all, the<br />
original RGB image (Figure 10 A) were transformed<br />
into 32-bit images, then we adjust the<br />
brightness/contrast and also applied smooth or find<br />
edges (Figure 10 B) option from processing images.<br />
The same image were analysed with CellC software<br />
(Fig. 10 C) to count the cells from filamentous<br />
cyanobacteria or to measure the size of each cells.<br />
A B<br />
C<br />
Fig 10. A – digital image of heterocystous<br />
cyanobacteria isolated from sulphurous mesothermal<br />
spring Obanul Mare (Mangalia) stained with AO; B –<br />
find edges of panel A using ImageJ software; C- total<br />
count analysis of panel A using CellC software (48<br />
cells counted from cyanobacteria’s filaments).<br />
Validation of any count were done using<br />
manual count.<br />
ImageJ software were used for<br />
automated measuring cell’s length (µm), using a<br />
133<br />
calibrated eyepiece graticule as reference (Ar<strong>de</strong>lean<br />
et al, 2009).<br />
Digital images from AO staining filaments of<br />
cyanobacteria in microcosm were treated with ImageJ<br />
to distinguish the heterocystous cell.<br />
Fig 11. Cyanobacteria with heterocyst presence in<br />
microcosm 2; AO staining (arrow indicate heterocyst<br />
cell present in samples of microcosm supplemented<br />
with gasoline).<br />
To avoid uncertain estimates of filament length<br />
and width, the number of filaments presented in one<br />
image should not be too high. Extreme filament<br />
<strong>de</strong>nsities would undoubtedly increase filament<br />
overlap and lead to uncertain measurements unless<br />
samples are diluted (Almesjö & Rolff, 2007).<br />
We use a blue light epifluorescence filter set to<br />
visualize AO-stained bacteria (N-400FL type). AO<br />
stains both DNA and RNA so is used for the<br />
enumeration of total bacteria.<br />
In figure 12 A we present only an example of<br />
digital analysis of fig.7 b: first, we adjust<br />
contrast/brightness of digital image, then analyse<br />
measure of graticula presented in fig. 7b and set the<br />
calibration bar to <strong>de</strong>termine correctly the length of<br />
each bacteria treated with nalidixic acid. In B is<br />
presented image analysis using CellC software.<br />
A B<br />
Fig 12. Image analysis program Image J (A) and<br />
CellC (B) from microcosm 2, elongated cells at time<br />
T4 (8hours)<br />
b) DAPI were used to view filamentous<br />
cyanobacteria isolated from mesothermal sulfurous
Utilization of epifluorescence microscopy…/ Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
spring and heterotrophic and phototrophic bacteria<br />
from marine environment.<br />
The fluorochrome DAPI is the most commonly<br />
bacterial stain for a wi<strong>de</strong> range of sample types.<br />
DAPI is a nonintercalating, DNA-specific stain which<br />
fluoresces blue or bluish-white (at or above 390 nm)<br />
when bound to DNA and excited with light at a<br />
wavelength of 365 nm (Kepner & Pratt, 1994). When<br />
unbound, or bound to non-DNA material, it may<br />
fluoresce over a range of yellow colors (see<br />
figure13). DAPI-stained filaments of cyanobacteria<br />
isolated from sulphurous spring Obanul Mare<br />
(Mangalia) reveal heterogeneous cells as can be seen<br />
in Fig.13a.<br />
a<br />
b<br />
Fig 13. DAPI-stained cyanobacteria isolated from<br />
sulphurous spring (a), arrows indicates septa between<br />
cells ; bacteria/cyanobacteria isolated from marine<br />
environment (b); both a and b treated with Image J<br />
and CellC software.<br />
Fig 14 . Cyanobacteria and heterotrophic cells in<br />
microcosm supplemented with gasoline/M2 - DAPI<br />
stain<br />
134<br />
c) Aniline blue is highly specific for staining<br />
type polysacchari<strong>de</strong>. Use of aniline blue is a good<br />
method not only for <strong>de</strong>tection of production of<br />
exocellular β-1,3-glucan, but also for <strong>de</strong>tection of<br />
some β-glucan in the cell wall (Nakanishi et al.,<br />
1976). In figure 15 is apparent the AB stained<br />
heterotrophic cells from microcosm supplemented<br />
with gasoline and cyanobacteria cells from<br />
microcosms and sulphurous spring samples.<br />
a b c<br />
Fig 15 . Visualisation of encapsulated bacteria and<br />
cyanobacteria after aniline-blue staining on M2 (a),<br />
M1 (b) and from sulphurous spring samples (c).<br />
d) PI staining is generally used for the<br />
evaluation of plasma membrane integrity by<br />
fluorescence. Literature mentions that molecular<br />
weighs PI is 668,4 and is thus assumed to be unable<br />
to penetrate cell membrane (Manini & Danovaro,<br />
2006). In figure16 living bacteria appeared green due<br />
to the excitation of the AO dye with which the cells<br />
have been stained and the samples stained with PI<br />
and appear red fluorescent cells; the bacteria were<br />
counted un<strong>de</strong>r blue excitation.<br />
a b<br />
c<br />
Fig 16. Marine bacteria examined using<br />
epifluorescence microscopy (magnification x1000),<br />
Fig (a) illustrate bacteria stained with propidium<br />
iodi<strong>de</strong> (<strong>de</strong>ad cells) in microcosms 1 and (b)<br />
respectively microcosms 2; total count analysis using<br />
the CellC software (c).
Simona Ghiţă et al. / Ovidius University Annals, Biology-Ecology Series 14: 127-137 (2010)<br />
e) Natural fluorescence - In figure 17 we<br />
<strong>de</strong>scribe succesfully separatation with ImageJ of cells<br />
by natural fluorescence of photosynthetic gasolinetolerant/oxidant<br />
microorganisms isolated from<br />
mesothermal sulphurous spring in different chanell –<br />
red and green- and then each image were automat<br />
counted with CellC, obtaining finally the number of<br />
cells red and green separately.<br />
Fig 17. Natural fluorescence of (A) photosynthetic<br />
gasoline-tolerant/oxidant microorganisms isolated<br />
from mesothermal sulphurous spring and digital<br />
image analysis of chlorophyll autofluorescence in (B)<br />
green channel ; (C) red channel and (D-E) total count<br />
analysis of red/green channels using the CellC<br />
software.<br />
In figure 18 there are presented images showing<br />
the natural fluorescence of chlorophyll, as an image<br />
of marine oxygenic gasoline tolerant/ oxidant<br />
phototrophic microorganisms. Difference is clearly<br />
apparent width of filaments of cyanobacteria<br />
<strong>de</strong>veloped in the experimental microcosms. The<br />
measurements were performed with Image J program<br />
as shown previous (see point 2).<br />
a b<br />
Fig 18. Autofluorescence of chlorophyll from<br />
oxygenic photosynthetic microorganisms:<br />
microcosms 1 (a) and 2 (b).<br />
Epifluorescence techniques and image analysis<br />
has increasingly been used to <strong>de</strong>termine cell size,<br />
bacterial abundance and <strong>de</strong>tection of physiological<br />
characteristics like damaged versus intact cell<br />
membranes.<br />
135<br />
Throughout the investigations conducted<br />
continuously attempted to <strong>de</strong>termine the nature of<br />
connections between communities of microorganisms<br />
and how and to which condition each.<br />
4. Conclusions<br />
The utilization of epifluorescence microscopy<br />
and digital image analysis enable us to study some<br />
morphological and functional aspects of prokaryotes:<br />
total cell counts (acridine orange, DAPI, SYBR green<br />
1), direct viable count (elongated cell in the presence<br />
of nalidixic acid, labelled with acridine orange),<br />
count of permeabilised cells (cells permeable to<br />
propidium iodi<strong>de</strong>), capsulated cell (labelled with<br />
aniline blue) and chlorophyll containing cells, both<br />
in enriched cultures and in natural / microcosms<br />
samples.<br />
The total number of heterotrophic cells counted<br />
using AO or DAPI is practically the same whereas<br />
total counts obtained with SYBR Green 1 are 47,6%<br />
higher.<br />
The number of <strong>de</strong>ad cells (PI positive) and that<br />
of (putative) capsulated cells are 12.3% and 10%,<br />
respectively of the total number ( AO and DAPI).<br />
The image analysis systems presented here was<br />
performed for counting and estimating the length of<br />
bacteria/cyanobacteria with uniform morphology.<br />
The presented methods does not totally exclu<strong>de</strong><br />
the need for manual microscope analyses of water<br />
samples, and automated procedures must<br />
intermittently be validated by in<strong>de</strong>pen<strong>de</strong>nt manual<br />
procedures.<br />
Acknowledgment<br />
We are grateful to Dr. Tech. Jyrki Selinummi<br />
(Department of Signal Processing, Tampere<br />
University of Technology, Finland) for very useful<br />
and kind advices concerning the use of software<br />
CellC and ImageJ.<br />
5. References<br />
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[2] MANINI E. & DANOVARO R., 2006- Synoptic<br />
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137
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
CHANGES IN BACTERIAL ABUNDANCE AND BIOMASS IN SANDY SEDIMENT<br />
MICROCOSMS SUPPLEMENTED WITH GASOLINE<br />
Dan Răzvan POPOVICIU 1 , Ioan ARDELEAN 1,2<br />
1 Ovidius University of Constanţa,Natural Sciences and Agricultural Sciences Faculty, Mamaia Avenue, no. 124,<br />
Constanţa, 900527, Romania, e-mail: dr_popoviciu@yahoo.com<br />
2 Biology Institute of Bucharest, Splaiul In<strong>de</strong>pen<strong>de</strong>nţei, no. 296, 060031 Bucureşti, Romania,<br />
email:ioan.ar<strong>de</strong>lean57@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: Bacterial abundance, biomass and morphological diversity were studied in three marine sediment<br />
microcosms: control, sediment supplemented with gasoline, and sediment supplemented with gasoline and<br />
ammonium nitrate. Microbial <strong>de</strong>nsity (4.7-6.65 × 10 6 cells/cm 3 sediment in uncontaminated samples) and<br />
biomass (1.72-3.13 µg/cm 3 sediment) dropped significantly after gasoline addition. Ammonium nitrate favoured<br />
a faster recovery to initial values. Gasoline contamination also modified the proportion of bacterial morphotypes,<br />
increasing the percentage of rod-shaped cells.<br />
Keywords: Bacteria, microcosms, hydrocarbons, abundance, biomass, morphotypes, sandy sediments.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Hydrocarbon contamination is one of the most<br />
frequent and most dangerous forms of pollution<br />
affecting marine environments. Studying its effects on<br />
prokaryote communities is important, both<br />
theoretically and practically, opening the way to<br />
bioremediation.<br />
From a microbiological point of view, sediments<br />
and not the water column are the richest marine<br />
environment. Both sandy and muddy sediments show<br />
significant amounts of prokaryotes, playing a key role<br />
in the <strong>de</strong>composition of organic matter and nutrient<br />
recycling..<br />
Abundance, biomass and composition of<br />
sediment bacterial communities can be <strong>de</strong>termined by<br />
many factors, such as the granulometric<br />
characteristics of the sediment, water dynamism,<br />
oxygenation, protozoan grazing etc [1, 2, 3].<br />
Even though they cover a large part of the marine<br />
littoral enviroment, coastal sands are the less studied<br />
[1].<br />
In or<strong>de</strong>r to evaluate the response of<br />
bacteriobenthos to various environmental changes,<br />
microcosms represent extremely valuable tools [4, 5].<br />
The objective of the present study was to<br />
<strong>de</strong>termine the effects of hydrocarbon addition on the<br />
abundance, biomass and morphological diversity of<br />
bacteria in marine sandy sediment microcosms.<br />
2. Material and Methods<br />
Microcosms. Sandy sediment was collected<br />
from the mediolittoral of a sandy beach in Constanţa,<br />
relatively close to the central headquarters of the<br />
“Ovidius” University, and wet sieved through a 2 mm<br />
sieve (in or<strong>de</strong>r to eliminate large particles and<br />
macrofauna) [6]. Three 1.4 L transparent plastic<br />
recipients were filled each with around 500 cm 3 of<br />
sand, covered by a 200 mL sea water column. All the<br />
microcosms were covered with transparent caps and<br />
stored at constant temperature (18°C) with<br />
illumination simulating the day-night cycle.<br />
The control microcosm was labeled “A”.<br />
Microcosm B was supplemented with 95 gasoline<br />
(1% final concentration). Microcosm C was<br />
supplemented with the same amount of gasoline, plus<br />
ammonium nitrate as a nutrient (0.005% final<br />
concentration).<br />
Sampling and fixation. Five samples consisting<br />
of sediment cores were collected from each<br />
microcosm at time intervals of 14 days. The first<br />
series of cores was taken just before the addition of<br />
gasoline and nutrient.<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010)<br />
Sample collection was done using improvised<br />
piston corers (20 mL syringes with the forepart<br />
<strong>de</strong>tached, but with the gradation intact). From each<br />
sample, the surficial 5 cm 3 (corresponding to a <strong>de</strong>pth<br />
of 17.5 mm) was taken for analysis.<br />
Each sample was suspen<strong>de</strong>d in 5 ml of buffered<br />
formaline (4% final concentration) [7, 8, 9, 10]. The<br />
formal<strong>de</strong>hy<strong>de</strong> solution acts as a fixative, killing the<br />
microorganisms and preventing contamination and<br />
cell <strong>de</strong>formation. The labeled tubes containing the<br />
subsamples were preserved by refrigeration at +4°C.<br />
Cell separation. Dislodgement of bacteria<br />
attached to sand grains is an important step prior to<br />
analysis. The procedure used was adapted, with some<br />
modifications, from existing literature [11, 12, 13, 14,<br />
15].<br />
Sediment suspensions were diluted 5-fold,<br />
incubated with Tween 80 (1 mg/mL final<br />
concentration) for 15 minutes and vortexed at 2 400<br />
r.p.m. for 5 minutes.<br />
Direct counting of bacteria. Microorganisms<br />
were visualised by epifluorescence microscopy, using<br />
3,6-dimethylaminoacridinic chlori<strong>de</strong> (acridine<br />
orange) as a fluorochrome. This compound becomes<br />
highly fluorescent by binding to the nucleic acids,<br />
giving an orange-red fluorescence for single-stran<strong>de</strong>d<br />
nucleic acids (mostly RNA) and a green one for<br />
double-stran<strong>de</strong>d acids (DNA) [16]. Acridine orange<br />
stains both living and <strong>de</strong>ad cells [17, 18].<br />
The technique employed was an adapted and<br />
simplified version of the protocols used by other<br />
authors [3, 8, 10, 11, 19, 20]. 1 ml was collected from<br />
each suspension and incubated for 5 minutes with 1<br />
ml acridine orange (5 µg/mL final concentration).<br />
The resulting solution was filtered through a 0.45 µm<br />
Millipore filtering membrane, using a syringe and a<br />
Millipore hol<strong>de</strong>r. Filtered membranes were<br />
previously stained with Sudan Black, in or<strong>de</strong>r to<br />
reduce background fluorescence.<br />
Each filter was washed with 50-60 ml of distilled<br />
water, placed on a glass sli<strong>de</strong> and examined using a<br />
Hund Wetzlar H 600 AFL 50 microscope, at an<br />
500× enlargement. An eyepiece grid micrometer was<br />
employed.<br />
For each filter, 15-20 grids were randomly<br />
chosen (from different areas of the membrane, except<br />
for its margins), photographed with a digital camera<br />
and visualised with MBF ImageJ for Microscopy<br />
140<br />
software (http://www.macbiophotonics.ca/downloads.<br />
htm.) [21].<br />
Fluorescent cells in each grid were counted<br />
manually. Fluorescent anorganic particles and<br />
obviously eukaryotic structures (by size and<br />
morphology) were exclu<strong>de</strong>d. In case sediment<br />
particles masked bacterial cells, any bacteria found<br />
on the surface of such particles were counted twice<br />
[7, 8, 17, 22]. The mean bacterial <strong>de</strong>nsity was<br />
calculated for each sample according to the following<br />
formula:<br />
N = n ×Af / Ag × V / v<br />
where:<br />
N = mean bacterial number per cm 3 of<br />
sediment;<br />
n = mean bacterial number per grid for each<br />
subsample;<br />
Ag = grid area;<br />
Af = filter area;<br />
v = volume of the filtered sediment<br />
suspension;<br />
V = volume of the total sediment suspension<br />
containing 1 cm 3 of sediment.<br />
Bacterial biomass estimation. All the<br />
microorganisms observed were classified into three<br />
morphological categories: cocci, bacilli (including<br />
coccobacilli and vibrios) and filamentous bacteria<br />
(those having a length more than five times greater<br />
than the width) [3]. Cell dimensions (diameter,<br />
respectively length and width) were measured using<br />
the grid micrometer.<br />
Biovolume was <strong>de</strong>termined for each cell<br />
according to the formula [8, 16]:<br />
V = (π/ 4) d 2 (l – d / 3)<br />
where:<br />
l = cell length;<br />
d = cell width/diameter.<br />
For cocci, the formula becomes:<br />
V = πd 3 / 6<br />
To <strong>de</strong>termine dry biomass based on the<br />
biovolume, several authors proposed different<br />
conversion factors. In the present study, the following<br />
formula was used [23]:<br />
md = 435 × V 0,86<br />
where:<br />
md = dry biomass (fg);
Dan Răzvan Popoviciu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010)<br />
V = cell volume (µm 3 ).<br />
Dry biomass was <strong>de</strong>termined for each cell,<br />
calculating then the media for each sample. Total<br />
(wet) biomass can be approximated using a<br />
conventional mean value for bacterial cell <strong>de</strong>nsity, of<br />
1.1 g/cm 3 [23, 24].<br />
3. Results and Discussions<br />
Bacterial cell abundance. The evolution of cell<br />
<strong>de</strong>nsity in time (from 0 to 56 days) for each<br />
microcosm is shown in Fig. 1.<br />
Million cells<br />
7<br />
6.5<br />
6<br />
5.5<br />
5<br />
4.5<br />
4<br />
3.5<br />
3<br />
0 14 28 42 56<br />
Time (days)<br />
A<br />
B<br />
C<br />
Fig. 1. Number of bacterial cells (× 10 6 ) per cm 3<br />
of sediment.<br />
For undisturbed sediment cores, bacterial <strong>de</strong>nsity<br />
ranged between 4.7-6.65 × 10 6 cells/cm 3 sediment<br />
with an average of 5.52 × 10 6 cells/cm 3 .<br />
These values are within the variation limits of<br />
littoral sediment microbial <strong>de</strong>nsity (although data<br />
found in literature is distributed over a wi<strong>de</strong> range).<br />
For comparison, here are some bacterial <strong>de</strong>nsities: 10 9<br />
cells/g dry sand [20], 5 × 10 8 -1.5 × 10 9 cells/g<br />
sediment [9] and 7-9 × 10 7 cells/g [25] on the U.S.A.<br />
East Coast, 1.91-7.32 × 10 7 cells/g dry sediment, in<br />
Eastern Canada, at the waterline [1], 3.6 × 10 8 cells/g<br />
dry sediment, in Florida [26], 6.8-20.3 × 10 8<br />
141<br />
cells/cm 3 , fine sands in a Mexican tropical lagoon, 1.2<br />
m <strong>de</strong>pth [27], 7 × 10 8 -6.7 × 10 9 cells/cm 3 , Baltic Sea<br />
[28], over 5.12 × 10 8 cells/g dry sediment, Western<br />
Mediterranean Sea [29], 1.5 × 10 8 cells/g dry sand<br />
[10], 6-8 × 10 9 cells/g sediment [30] and 3.54-8.08 ×<br />
10 9 cells/g [3] in the Adriatic Sea, at several meters<br />
<strong>de</strong>pth, 0.2-1 × 10 9 cells/g dry sediment, in littoral<br />
sands in the Gulf of Tokyo [14], 2.56-4.46 × 10 6<br />
cells/g, at 2 m <strong>de</strong>pth, in North Sea [31].<br />
The addition of gasoline caused a <strong>de</strong>crease in cell<br />
abundance to values as low as 3.6 × 10 6 cells/cm 3 . A<br />
return to <strong>de</strong>nsities similar to the initial ones was<br />
observed in the last samples. The recovery was faster<br />
in the microcosm supplemented with ammonium<br />
nitrate (28 days).<br />
Direct cuantification of bacteria through<br />
epifluorescence microscopy has some limitations.<br />
Cell masking by sediment particles, background<br />
fluorescence, lack of an efficient method to<br />
distinguish prokaryotes from eukaryotes, the poor<br />
quality of some photographs etc., can cause<br />
overestimation or un<strong>de</strong>restimation of real abundance<br />
[8, 17, 22]. The method used for bacterial dispersion<br />
from sediment grains can also influence the results<br />
[9].<br />
An important factor that can cause<br />
un<strong>de</strong>restimation of bacterial abundance is the<br />
extremely small size of some microorganisms. Many<br />
bacteria have diameters below 0.3 microns, and can<br />
be very difficult or even impossible to visualise,<br />
<strong>de</strong>pending on the optical means employed. Some of<br />
them can even pass through usual filtering<br />
membranes. According to some authors such<br />
ultramicrobacteria constitute up to 72% of the soil<br />
microbiota, and it seems that they have similar<br />
proportions in marine environments [17]. In<br />
conclusion, all data obtained using direct counts<br />
should be regar<strong>de</strong>d as relative.<br />
It should be noted that not all the bacteria<br />
ennumerated with acridine orange are alive. Living<br />
bacteria constitute usually less than one third, rarely<br />
reaching 60% of the total number. The rest are <strong>de</strong>ad<br />
cells, or even cell fragments [10, 32].
Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010)<br />
Biomass (µg)<br />
3.5<br />
3<br />
2.5<br />
2<br />
1.5<br />
1<br />
0 14 28 42 56<br />
Time (days)<br />
Fig. 2. Bacterial dry biomass (µg/cm 3 ).<br />
Bacterial biomass<br />
Biomass showed large variations, from 1.36 to<br />
3.13 µg/cm 3 sediment (equiv<strong>ale</strong>nt to 4.3 to 11.4 µg<br />
total biomass/cm 3 ). On average, the highest biomass<br />
was <strong>de</strong>termined for the undisturbed sediment (an<br />
average of 2.27 µg/cm 3 ). The addition of gasoline was<br />
followed by a <strong>de</strong>crease in microcosms B and C. The<br />
average value for contaminated sediment in<br />
microcosm B was only 1.7 µg/cm 3 , while in C, it was<br />
higher (2.12 µg/cm 3 ), showing a faster recovery.<br />
The importance of nitrogenous nutrients in the<br />
recovery of natural microbiota after hydrocarbon<br />
pollution is consistent with data in existing literature<br />
[33, 34].<br />
The exact <strong>de</strong>termination of bacterial biomass can<br />
be affected by various technical and mathematical<br />
factors. Different fluorochromes can give different<br />
results [16]. The selected biovolume to biomass<br />
conversion factor influences the final results. Also, it<br />
was <strong>de</strong>monstrated that coastal marine sediments<br />
contain significant numbers of disk-shaped bacteria<br />
and counting them as cocci would overestimate their<br />
volume [35].<br />
Proportion of major bacterial morphotypes.<br />
As specified above, bacteria were classified into three<br />
groups: cocci, rods and filamentous. Their proportion<br />
in the total abundance, for each microcosm and<br />
collection time is shown in Fig. 3 (a,b,c).<br />
A<br />
B<br />
C<br />
142<br />
100%<br />
80%<br />
60%<br />
40%<br />
20%<br />
0%<br />
100%<br />
80%<br />
60%<br />
40%<br />
20%<br />
0%<br />
100%<br />
80%<br />
60%<br />
40%<br />
20%<br />
0%<br />
Microcosm A<br />
0 14 28 42 56<br />
Time (days)<br />
Microcosm B<br />
0 14 28 42 56<br />
Time (days)<br />
Microcosm C<br />
0 14 28 42 56<br />
Time (days)<br />
Filamentous<br />
Rods<br />
Cocci<br />
Filamentous<br />
Rods<br />
Cocci<br />
Filamentous<br />
Rods<br />
Cocci<br />
Fig.3 (a,b,c). Percentage of major bacterial<br />
morphotypes
Dan Răzvan Popoviciu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010)<br />
Most of the bacterial cells (72-92%) observed<br />
were spherical (in accordance to data obtained in<br />
a)<br />
a)<br />
b)<br />
c)<br />
Fig. 4. Main bacterial morphotypes: a) cocci; b)<br />
rods; c) filamentous (bar = 10 µm)<br />
143<br />
marine sediments by Šestanović et al. [3] and<br />
Popoviciu [36]).<br />
The proportion of rod-shaped bacteria (including<br />
coccobacilli and vibrios) was different among the<br />
three microcosms. In undisturbed sediment, their<br />
percentage was generally below 15% (note: in<br />
microcosm A, at T3, the high percentage was due to a<br />
single large colony of small rods), with an average of<br />
13.9%. In gasoline contaminated sediment, rodshaped<br />
bacteria constituted a larger part of the<br />
microbiota, with an average of 23.4%. The<br />
proportion of filamentous bacteria was insignificant.<br />
Bacterial assemblages were rare, in concordance<br />
to the observations ma<strong>de</strong> by Novitsky & MacSween<br />
[1].<br />
It should be noted that classification of small<br />
bacteria (cells with diameters below 0.6 microns<br />
formed the majority) into morphotypes is prone to<br />
errors. This is due to the fluorescent halo that appears<br />
around cells, causing very small sized bacilli or<br />
vibrios to be counted as cocci [19].<br />
4. Conclusions<br />
Hydrocarbon contamination affects marine<br />
sediment microbiota in terms of abundance, biomass<br />
and composition.<br />
Addition of nitrogenous nutrients (ammonium<br />
nitrate) favours a faster recovery to initial parameters.<br />
Epifluorescence microscopy is a useful tool for<br />
evaluating the reaction of sediment bacteria to<br />
environmental changes. In perspective, use of<br />
differential fluorochromes and correlation to<br />
cultivation techniques are to be employed in such<br />
studies.<br />
5. References<br />
[1] NOVITSKY, J.A., MACSWEEN, M.C., 1989 –<br />
Microbiology of a high energy beach sediment:<br />
evi<strong>de</strong>nce for an active and growing community.<br />
Mar. Ecol. Prog. Ser. 52: 71-75.<br />
[2] BRUNE, A., FRENZEL, P., CYPIONKA, H.,<br />
2000 – Life at the oxic-anoxic interface:<br />
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[3] ŠESTANOVIĆ, S., SOLIĆ, M.,<br />
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[4] RÖLING, W.F.M., MILNER, M.G., JONES,<br />
D.M., LEE, K., DANIEL, F., SWANNELL,<br />
R.J.P., HEAD, I.M., 2002 – Robust<br />
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[5] MIRALLES, G., NÉRINI, D., MANTÉ, C.,<br />
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MICHOTEY, V., NAZARET, S., BERTRAND,<br />
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HOPKINSON, C.S., 1983 – Bacterial<br />
production in marine sediments: will cell-<br />
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metabolism? Mar. Ecol. Prog. Ser. 11: 119-127.<br />
[12] ELLERY, W.N., SCHLEYER, M.H., 1984 –<br />
Comparison of homogenization and<br />
ultrasonication as techniques in extracting<br />
attached sedimentary bacteria. Mar. Ecol. Prog.<br />
Ser. 15: 247-250.<br />
[13] EPSTEIN, S.S., ROSSEL, J., 1995 –<br />
Enumeration of sandy sediment bacteria: search<br />
for optimal protocol. Mar. Ecol. Prog. Ser 117:<br />
289-298.<br />
[14] KUWAE, T., HOSOKAWA, Y., 1999 –<br />
Determination of abundance and biovolume of<br />
bacteria in sediments by dual staining with 4’,6diamidino-2-phenylindole<br />
and acridine orange:<br />
relationship to dispersion treatment and<br />
sediment characteristics. Appl. Environ.<br />
Microbiol. 65: 3407-3412.<br />
[15] BENNETT, P.C., ENGEL, A.S., ROBERTS,<br />
J.A., 2006 – Counting and imaging bacteria on<br />
mineral surfaces. in Patricia, J., Maurice, A.,<br />
Warren, L.A. (eds.). Methods of Investigating<br />
Microbial-Mineral Interactions. CMS Workshop<br />
Lectures, Vol. 14: 37-78, The Clay Mineral<br />
Society, Chantilly.<br />
[16] SHERR, B., SHERR, E., DEL GIORGIO, P.,<br />
2001 – Enumeration of total and highly active<br />
bacteria. Meth. Microbiol. 30: 129-159.<br />
[17] KEPNER, R.L., PRATT, J.R., 1994 – Use of<br />
fluorochromes for direct enumeration of total<br />
bacteria in environmental samples: past and<br />
present. Microbiol. Rev. 58: 603-615.<br />
[18] MCFETERS, G.A., YU, F.P., PYLE, B.H.,<br />
STEWART, P.S., 1995 – Physiological<br />
assessment of bacteria using fluorochromes. J.<br />
Microbiol. Meth. 21: 1-13.<br />
[19] WATSON, S.W., NOVITSKY, T.J., QUINBY,<br />
H.L., VALOIS, F.W., 1977 – Determination of<br />
bacterial number and biomass in the marine<br />
environment. Appl. Environ. Microbiol. 33:<br />
940-946.<br />
[20] MONTAGNA, P.A., 1982 – Sampling <strong>de</strong>sign<br />
and enumeration statistics for bacteria extracted<br />
from marine sediments. Appl. Environ.<br />
Microbiol. 43: 1366-1372.<br />
[21] COLLINS, T.J., 2007 – ImageJ for microscopy.<br />
BioTechniques 43: 25-30.
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[22] GOUGH, H.L., STAHL, D.A., 2003 –<br />
Optimization of direct cell counting in sediment.<br />
J. Microbiol. Meth. 52: 39-46.<br />
[23] LOFERER-KRÖßBACHER, M., KLIMA, J.,<br />
PSENNER, R., 1998 – Determination of<br />
bacterial cell dry mass by transmission electron<br />
microscopy and <strong>de</strong>nsitometric image analysis.<br />
Appl. Environ. Microbiol. 64: 688-694.<br />
[24] BAKKEN, L.R., OLSEN, R.A., 1983 – Buoyant<br />
<strong>de</strong>nsities and dry-matter contents of<br />
microorganisms: conversion of a measured<br />
biovolume into biomass. Appl. Environ.<br />
Microbiol. 45: 1188-1195.<br />
[25] HYMEL, S.N., PLANTE, C.J., 1998 –<br />
Improved method of bacterial enumeration in<br />
sandy and <strong>de</strong>posit-fee<strong>de</strong>r gut sediments using<br />
the fluorescent stain 4,6-diamidino-2phenylindole<br />
(DAPI). Mar. Ecol. Prog. Ser.<br />
173: 299-304.<br />
[26] PROCTOR, L.M., SOUZA, A.C., 2001 –<br />
Method for enumeration of 5-cyano-3,2-ditoyl<br />
tetrazolium chlori<strong>de</strong> (CTC)- active cells and<br />
cell-specific CTC activity of benthic bacteria in<br />
riverine, estuarine and coastal sediments. J.<br />
Microbiol. Meth. 43: 213-222.<br />
[27] FERRARA-GUERRERO, M.J.,<br />
CASTELLANOS-PAÉZ, M.E., GARZA-<br />
MOURIÑO, G., 2007 – Variation of a benthic<br />
heterotrophic bacteria community with different<br />
respiratory metabolisms in Coyuca <strong>de</strong> Benitez<br />
coastal lagoon (Guerrero, Mexico). Rev. Biol.<br />
Trop. (Int. J. Trop. Biol.) 55: 157-169.<br />
[28] DIETRICH, D., ARNDT, H., 2000 – Biomass<br />
partitioning of benthic microbes in a Baltic<br />
inlet: relationships between bacteria, algae,<br />
heterotrophic flagellates and ciliates. Mar. Biol.<br />
136: 309-322.<br />
[29] DANOVARO, R., FABIANO, M., BOYER,<br />
M., 1994 – Seasonal changes of benthic bacteria<br />
in a seagrass bed (Posidonia oceanica) of the<br />
Ligurian Sea in relation to origin, composition<br />
and fate of the sediment organic matter. Mar.<br />
Biol. 119: 489-500.<br />
[30] PUSCEDDU, A., FIORDELMONDO, C.,<br />
DANOVARO, R., 2005 – Sediment<br />
resuspension effects on the benthic microbial<br />
loop in experimental microcosms. Microb. Ecol.<br />
50: 602-613.<br />
145<br />
[31] LUNAU, M., LEMKE, A., WALTHER, K.,<br />
2005 – An improved method for counting<br />
bacteria from sediments and turbid<br />
environments by epifluorescence microscopy.<br />
Environ. Microbiol. 7: 961-968.<br />
[32] ZWEIFEL, U.L., HAGSTRÖM, Å., 1995 –<br />
Total counts of marine bacteria inclu<strong>de</strong> a large<br />
fraction of non-nucleoid-containing bacteria<br />
(ghosts). Appl. Environ. Microbiol. 61: 2180-<br />
2185.<br />
[33] HIGASHIHARA, T., SATO, A., SIMIDU, U.,<br />
1978 – An MPN method for the enumeration of<br />
marine hydrocarbon <strong>de</strong>grading bacteria. Bull.<br />
Japan. Soc. Sci. Fish. 44: 1127-1134.<br />
[34] HAZEN, T.C., 2010 – Biostimulation, in<br />
Timmis, K.N. (ed.). Handbook of Hydrocarbon<br />
and Lipid Microbiology, 4517-4530, Springer-<br />
Verlag Berlin Hei<strong>de</strong>lberg.<br />
[35] MUDRYK, Z.J., PODGÓRSKA, B., 2006 –<br />
Scanning electron microscopy investigation of<br />
bacterial colonization of marine beach sand<br />
grains. Baltic Coastal Zone 10: 61-72.<br />
[36] POPOVICIU, D.R., 2009 – Aspecte cantitative<br />
<strong>şi</strong> morfo-structur<strong>ale</strong> <strong>ale</strong> microbiotei din<br />
sedimente marine nisipoase <strong>de</strong> la litoralul<br />
românesc al Mării Negre. Master’s thesis,<br />
Ovidius University of Constanţa, Faculty of<br />
Natural Sciences and Agricultural Sciences.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE FORMATION OF BACTERIAL BIOFILMS ON THE HYDROPHILE<br />
SURFACE OF GLASS IN LABORATORY STATIC CONDITIONS: THE EFFECT<br />
OF TEMPERATURE AND SALINITY<br />
Aurelia Manuela MOLDOVEANU *, Ioan I. ARDELEAN **<br />
* Ovidius” University Constanta, 1Universitatii Alley, Building B, 900527 Constanta, Romania,<br />
aurelia.moldoveanu@yahoo.com<br />
** Biology Institute of Bucharest, 296 Splaiul In<strong>de</strong>pen<strong>de</strong>nţei, 060031 Bucharest, Romania,<br />
ioan.ar<strong>de</strong>lean57@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: In the case of temperature variation, at 18ºC there is an increase of the cellular <strong>de</strong>nsity from<br />
12∙10 2 cel/mm 2 to 62∙10 2 cel/mm 2 , while at 6 ºC cellular <strong>de</strong>nsity increases from 5∙10 2 cel/mm 2 to 55∙10 2<br />
cel/mm 2 . The results obtained show that cellular <strong>de</strong>nsity in the case of biofilms formed at 6 ºC is lower<br />
compared to cellular <strong>de</strong>nsity of biofilms formed at 18 ºC. Salinity modification from 15g/l to 10g/l<br />
<strong>de</strong>termined an increase of cellular <strong>de</strong>nsity from 4∙10 2 cel/mm 2 to 54∙10 2 cel/mm 2 , while the modifications<br />
of the osmotic conditions in the marine environment due to salinity <strong>de</strong>crease to 5g/l led to an increase of<br />
the cellular <strong>de</strong>nsity from 2∙10 2 cel/mm 2 to 49∙10 2 cel/mm 2 . The variation of temperature and salinity of<br />
seawater in “in vitro” conditions influenced the process of bacterial adherence and formation of the initial<br />
layers of the biofilms by the modification of the <strong>de</strong>nsity of the adherent cells.<br />
Keywords: quorum sensing, exopolysacchari<strong>de</strong>s, matrix, microecosystem, microfouling.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Biofilms are complex structures ma<strong>de</strong> up of<br />
cells and exopolysacchari<strong>de</strong>s which form at the level<br />
of interfaces and which are intensely studied because<br />
of their fundamental importance and applicability in<br />
the environmental domain, biotechnology and<br />
medicine [1,2,3,4,5].<br />
Marine bacteria form biofilms in “in situ”<br />
conditions, un<strong>de</strong>r the influence of various<br />
environmental factors. Hydrostatic pressure, solar<br />
radiation, temperature, salinity, pH, oxidation<br />
potential and nutrients existing on the surfaces are<br />
physic-chemical factors that influence the activity of<br />
microorganisms, but their role on the marine bacterial<br />
populations is still being studied [6,7,5]. Among<br />
these factors, temperature and salinity have major<br />
importance for all living organisms, especially for<br />
those in the marine environment, where<br />
microorganisms are subjected to extremely wi<strong>de</strong><br />
variations which allowed them to survive from the<br />
beginning of life on Earth. They are the only<br />
organisms that can adapt to extreme environments<br />
[8,9,10].<br />
In laboratory conditions, the variation of<br />
environmental factors is essential in the formation of<br />
biofilms. It is important to know which factor has the<br />
most influence on the adherent bacterial cells. Thus,<br />
the growth and multiplication of microorganisms is<br />
the result of a number of coordinated metabolic<br />
reactions whose normal <strong>de</strong>velopment is ensured by an<br />
optimal temperature [11,12,13].<br />
The marine bacteria in the structure of biofilms<br />
react to temperature and modify their bacterial<br />
metabolism and the mechanism for the regulation of<br />
genes <strong>de</strong>pending on how this factor varies, most<br />
species being studied between their optimal<br />
temperature limits due to the mesophile character<br />
[14]. Thus, an increase by 10 ºC of the initial<br />
temperature <strong>de</strong>termines an increase of the speed of<br />
the chemical reactions and gene regulation<br />
mechanism. Consequently, the speed of the enzymatic<br />
processes increases progressively as the temperature<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
rises until it reaches the optimum level and then the<br />
speed <strong>de</strong>creases progressively [15].<br />
The natural environments offer microorganisms<br />
different conditions of salinity, from very low<br />
concentrations (rivers and lakes) to the very high<br />
concentrations of salty lakes and seas, or even to<br />
those that represent true saturated solutions. Thus,<br />
salinity becomes a variable factor in the marine<br />
environment which is important for the<br />
microorganisms within the biofilms as they are<br />
influenced by it according to the <strong>de</strong>gree of tolerance<br />
to the concentration of NaCl and the mechanism for<br />
the regulation of the available ions [16]. The study of<br />
the effect of temperature and salinity on the temporal<br />
dynamics of bacterial cell <strong>de</strong>nsity on the hydrophile<br />
surface of glass in laboratory static conditions leads<br />
to personal data regarding the initial stages of biofilm<br />
formation.<br />
2. Material and Methods<br />
In our experiments, we used two static methods<br />
in or<strong>de</strong>r to <strong>de</strong>termine the environmental factors with<br />
role in biofilm formation: the Henrici method<br />
[17,18,19], wi<strong>de</strong>ly employed in the study of<br />
adherence and the microbial fishing method [20,21],<br />
a more recent adaptation of the classical method.<br />
The surfaces were subjected to a sterilization<br />
process in or<strong>de</strong>r to diminish the possible<br />
contamination with microorganisms of the glass<br />
sli<strong>de</strong>s which will serve as support for the adherent<br />
marine bacteria. The sli<strong>de</strong>s were <strong>de</strong>greased with<br />
ethanol 70% and sterilized in the drying oven at 180<br />
ºC for one hour [22].<br />
In or<strong>de</strong>r to obtain biofilms, two types of liquid<br />
culture media were used: seawater from the littoral<br />
zone and seawater kept in aquarium conditions in the<br />
Laboratory for Biodiversity Investigation within<br />
“Ovidius” University of Constanta. The aquarium<br />
seawater is frequently used in the study of biofilms<br />
and marine microfouling and it was used in or<strong>de</strong>r to<br />
observe the possible facilitation of their formation<br />
[23].<br />
The method used is accomplished in static<br />
conditions in sterile containers in which 100 ml of<br />
seawater were poured and the sli<strong>de</strong>s were introduced.<br />
This type of method is more advantageous for the<br />
formation of biofilms when there is no system for<br />
148<br />
water recirculation, according to [24], who claims<br />
that the methods with continuous flux prevent the<br />
rapid formation of biofilms within the first hours.<br />
The support sli<strong>de</strong>s for the adherent bacteria<br />
were positioned according to the mentioned methods<br />
in an inclined position compared to the classical<br />
method, in or<strong>de</strong>r to avoid the sedimentation<br />
phenomenon which <strong>de</strong>termines the occurrence of<br />
high <strong>de</strong>nsities of the adherent marine bacteria [25].<br />
The experiment was accomplished in a<br />
thermostatic room at a constant temperature of 18 ºC<br />
in the Laboratory for Biodiversity Investigation<br />
within “Ovidius” University of Constanta and in a<br />
refrigerator at a constant temperature of 6 ºC. The<br />
salinity modification was done only for the littoral<br />
seawater and not for the aquarium water which is a<br />
microecosystem. Thus, seawater salinity, which has a<br />
normal value of 15g/l was modified by adding<br />
osmosis water and certain mixtures per liter obtaining<br />
thus two experimental versions: in the first version,<br />
normal salinity was <strong>de</strong>creased to 10g/l by adding<br />
333ml of osmosis water in 666ml of seawater; in the<br />
second version a salinity of 5g/l was obtained by<br />
adding 666 ml of osmosis water in 333 ml of<br />
seawater.<br />
The study of biofilms was accomplished over a<br />
period of 36 hours during which there was an interval<br />
when no samples were collected. Sample collection<br />
occurred for 12 hours in the first day hourly, followed<br />
by an interval of 12 hours when no samples were<br />
collected and again the following day samples were<br />
collected every two hours for 12 hours.<br />
After collection the sli<strong>de</strong>s were subjected to a<br />
process of fixation with 2.5% formal<strong>de</strong>hy<strong>de</strong> solution<br />
in artificial seawater (solution with marine salts with<br />
a concentration of 18g/l, similar to the Black Sea) for<br />
30 minutes and then subjected to <strong>de</strong>salinization by<br />
washing for 10 minutes in three successive solutions<br />
with the following content: 75% artificial seawater<br />
with 25% osmosis water, 50% artificial seawater with<br />
50% osmosis water and 100% osmosis water. The<br />
<strong>de</strong>salinization was realized in or<strong>de</strong>r to prevent the<br />
formation of salt crystals which absorb the<br />
fluorescent coloring matter and reflect it, affecting<br />
thus the cell visualization [26].<br />
After <strong>de</strong>salinization, the samples were<br />
introduced in a solution with 0.5% gentian violet in<br />
10 ml ethanol and 90 ml distilled water for one
Aurelia Manuela Moldoveanu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
minute. Afterwards, they were abundantly washed<br />
twice with osmosis water in or<strong>de</strong>r to eliminate the<br />
excess of coloring matter [27].<br />
The sample investigation was done by means of<br />
the Hund microscope, cell counting being done using<br />
an ocular grid, calibrated according to the standard<br />
procedure [28]; cells within 20 microscopic fields<br />
per each sample were counted, according to the<br />
standard counting procedures for the surface bacteria<br />
[29]. Thus, the values of cellular <strong>de</strong>nsity are<br />
expressed on the graphs represented by the mean of<br />
the 80 microscopic fields per each sample.<br />
3. Results and Discussions<br />
3.1 The chemical analysis of water<br />
There are differences between the two types of<br />
culture media used for the generation of bacterial<br />
biofilms on the hydrophile surface of glass sli<strong>de</strong>s and<br />
in or<strong>de</strong>r to emphasize their existence we analyzed the<br />
seawater samples in the Chemistry Laboratory within<br />
the “Grigore Antipa” Marine Research Institute of<br />
Constanta (Table 1).<br />
The chemical analysis of seawater emphasized<br />
the existence of differences among the chemical<br />
parameters: salinity, pH, concentration of inorganic<br />
substances between the two types of seawater used.<br />
The littoral seawater has normal parameters also<br />
registered in previous years [30], but the aquarium<br />
seawater has values well over the normal limit with<br />
an increase of over 10g/l of salinity and a <strong>de</strong>crease of<br />
pH from 8.12 (normal value for littoral seawater) to<br />
6.56 units for the aquarium water, almost two units<br />
less than the initial value. The concentration of<br />
inorganic substances is well above the normal one for<br />
seawater. The concentration of nitrates is three times<br />
higher compared to the normal value, while the<br />
concentration of polyphosphates is over 84 times<br />
higher.<br />
The existence of these differences between the<br />
two used culture media can cause changes in the<br />
formation manner of bacterial biofilms in liquid<br />
medium, as well as the temporal dynamic of their<br />
formation. The bacterial biofilms formed are an<br />
assemblage of surface-associated microbial cells that<br />
149<br />
is enclosed in an extracellular polymeric substance<br />
matrix.<br />
3.2 The formation of biofilms un<strong>de</strong>r the<br />
influence of temperature<br />
Figure one shows the values of cellular <strong>de</strong>nsity<br />
obtained after the modification of the temperature<br />
factor for the biofilms formed on the hydrophile<br />
surface of glass sli<strong>de</strong>s and collected from the<br />
containers with littoral seawater kept at a constant<br />
temperature of 18ºC and 6 ºC, respectively.<br />
The data analysis emphasized the existence of<br />
successive stages for the formation of biofilms. Thus,<br />
in the case of the biofilms formed at 18 ºC, one hour<br />
after the sli<strong>de</strong>s immersion the cellular <strong>de</strong>nsity is<br />
12∙10 2 cel/mm 2 . This value doubles eight hours later<br />
to 25 ∙10 2 cel/mm 2 and increases progressively to a<br />
ten<strong>de</strong>ncy to triple the cellular <strong>de</strong>nsity to 37 ∙ 10 2<br />
cel/mm 2 11 hours later. After 12 hours, during which<br />
the sli<strong>de</strong>s were left over night, the following day the<br />
cellular <strong>de</strong>nsity reaches the value of 45∙10 2 cel/mm 2<br />
24 hours after immersion. The value increases<br />
progressively to 60∙10 2 cel/mm 2 36 hours after<br />
immersion<br />
For the seawater in the containers kept at 6 ºC in<br />
the refrigerator, there is a progressive increase from 5<br />
∙10 2 cel/mm 2 only one hour after immersion and a<br />
doubling of this value seven hours later to 10 ∙10 2<br />
cel/mm 2 , as well as tripling to 15∙10 2 cel/mm 2 eight<br />
hours later. The following day, after 12 hours, the<br />
cellular <strong>de</strong>nsity was 41∙10 2 cel/mm 2 and increased<br />
progressively to 54∙10 2 cel/mm 2 .<br />
The progression of cellular <strong>de</strong>nsity growth is<br />
over 2.3 for the biofilms formed at 18 ºC during the<br />
first 12 hours and below 1.2 after 24 hours. Also, in<br />
the case of the biofilms formed in containers kept at 6<br />
ºC, the progression is over 1.9 during the first 12<br />
hours and below 1.1 after 24 hours. On the first day,<br />
after 12 hours, there is a difference of approx. 9∙10 2<br />
cel/mm 2 between the two progressions of <strong>de</strong>nsity<br />
growth, <strong>de</strong>pending on temperature. 24 hours later, the<br />
difference is below 8∙10 2 cel/mm 2 and 36 hours later<br />
it rises to 10∙10 2 cel/mm 2 .<br />
A number of experiments regarding bacterial<br />
adhesion were accomplished on different types of<br />
surfaces (copper, PVC and polybuten) by Rogers [31]<br />
at different temperatures (20 ºC, 40 ºC, 50 ºC and 60
The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
ºC) using the species Legionella pneumophila (a<br />
species with wi<strong>de</strong> temperature limits between 5.7 and<br />
63 ºC) and other strains of non-Legionella type over a<br />
period of 21 days. As a result, it was observed that<br />
the used strains displayed a logarithmic growth with a<br />
<strong>de</strong>nsity value of 1.3∙10 4 cel/cm 2 in the growing phase<br />
and 7.56∙10 4 cel/cm 2 on polybutylene and PVC<br />
surfaces for the non-Legionella strains at 20 ºC and<br />
4.25∙10 4 cel/cm 2 for polybutylene surface at 60 ºC. It<br />
is evi<strong>de</strong>nt that the colonization is higher on the<br />
hydrophobe surfaces at 20 ºC, compared to 60 ºC<br />
when the number of bacteria <strong>de</strong>creases due to the<br />
exceeding of the optimal temperature for<br />
microorganism <strong>de</strong>velopment.<br />
Experiments regarding the colonization of<br />
surfaces by the bacterium B<strong>de</strong>llovibrio bacteriovorus<br />
were accomplished by Kelley [32] un<strong>de</strong>r the<br />
influence of different temperatures (between 4 and 29<br />
ºC), using clam valves, glass and polystyrene as<br />
substrate, and observing the existence of positive<br />
correlations in the case of the factor temperature and<br />
the formation of biofilms, with maximum association<br />
of cells in the biofilms at 18 ºC and a minimum one at<br />
14 ºC, as well as a significant <strong>de</strong>crease of <strong>de</strong>nsity at<br />
temperatures below 5 ºC after 24 hours, followed by<br />
a progressive increase of <strong>de</strong>nsity 120 hours after the<br />
beginning of the experiment.<br />
The values obtained <strong>de</strong>monstrated a logarithmic<br />
increase of the number of adherent cells from 1.1 ∙10 5<br />
CFU/cm 2 to 1.4∙10 5 CFU/cm 2 for the clam valves,<br />
1.7∙10 3 CFU/cm 2 and 1.8∙10 4 CFU/cm 2 for glass<br />
and 5.4∙10 3 CFU/cm 2 and 1.0 ∙10 4 CFU/cm 2 for<br />
polystyrene.<br />
A number of experiments regarding the capacity<br />
of accomplished certain isolates of<br />
Stenotrophomonas maltophilia to form biofilms in<br />
variable temperature conditions (18 ºC, 32 ºC, 37 ºC)<br />
by were realized by Di Bonaventura [33] using<br />
different strains. There is an increase of the quantity<br />
of biofilms for the strains exposed to 32 ºC after one<br />
day to 0.680 BPI compared to those exposed to 18 ºC<br />
(0.557 BPI) and 37 ºC (0.491 BPI). In what regards<br />
the used strains, the temperature did not modify<br />
significantly their distribution: 82% of those used<br />
formed biofilms and only 2% did not form them. One<br />
strain formed biofilms only at 18 ºC and two strains<br />
only at 32 ºC. The capacity to forms biofilms is<br />
150<br />
important even at room temperature (18 ºC), but the<br />
adherence value is lower.<br />
The following day, after the 12 hour interval<br />
when no samples were collected, the <strong>de</strong>nsity value<br />
was 41∙10 2 cel/mm 2 and there was a progressive<br />
growth towards 55∙10 2 cel/mm 2 .<br />
Data in specialized literature confirm the<br />
existence of a growth in bacterial <strong>de</strong>nsity <strong>de</strong>pending<br />
on the exposure time of the surfaces to aquatic<br />
environment and the increase of temperature. Thus, at<br />
18 ºC, up to 25-30 ºC, there is an optimal bacterial<br />
growth. But temperatures over 35 ºC, 40 ºC and 50<br />
ºC affect the formation of biofilms because the<br />
optimum limits for the survival of certain bacterial<br />
species are excee<strong>de</strong>d<br />
Our experiments took place between the<br />
optimum limits for mesophile bacteria, noticing an<br />
increase of the <strong>de</strong>nsity values at 18 ºC, compared to 6<br />
ºC (kept in a refrigerator).<br />
In the case of the sli<strong>de</strong>s immersed in containers<br />
with aquarium water at 18 ºC, Figure 2 displays an<br />
increase of the bacterial <strong>de</strong>nsity from 16∙10 2 cel/mm 2<br />
one hour after immersion to a double value of 32∙10 2<br />
cel/mm 2 eight hours later. After 12 hours, during<br />
which the sli<strong>de</strong>s were left over night, there is a<br />
ten<strong>de</strong>ncy for the tripling of the cellular <strong>de</strong>nsity to<br />
49∙10 2 cel/mm 2 22 hours after the immersion of the<br />
sli<strong>de</strong>s into liquid medium and a progressive increase<br />
towards 62∙10 2 cel/mm 2 .<br />
For the containers kept at 6 ºC, the <strong>de</strong>nsity<br />
increases to 5∙10 2 cel/mm 2 one hour from immersion<br />
towards a double value of 13∙10 2 cel/mm 2 seven<br />
hours later and a progressive growth from 22 ∙10 2<br />
cel/mm 2 ten hours later when there is a ten<strong>de</strong>ncy for<br />
a triple value of the <strong>de</strong>nsity of adherent bacteria.<br />
The growth occurs based on a progression of<br />
2.5 in the case of biofilms formed in aquarium water<br />
kept at 18 ºC during the first 12 hours. There is a<br />
<strong>de</strong>crease to 1.1 after 24 hours from immersion. The<br />
difference between the two progressions is 4∙10 2<br />
cel/mm 2 during the first 12 hours from immersion<br />
and it increases to 8 ∙10 2 cel/mm 2 24 hours later. It<br />
<strong>de</strong>creases 36 hours later to 7 ∙10 2 cel/mm 2 .<br />
In variable conditions of temperature, the<br />
bacterial colonization occurs more quickly in<br />
aquarium seawater. Thus, Toren [34] realizes<br />
experiments regarding the formation of biofilms<br />
(Vibrio sp. strain AK-1) on a coral surface in case of
Aurelia Manuela Moldoveanu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
temperature variation (16º C, 23º C, 29º C) and<br />
registers a <strong>de</strong>crease of the quantity of inoculate from<br />
1.2 ∙10 8 cel/l to 1.2∙10 2 cel/l used with the increase of<br />
temperature, as well as an increased adhesion at high<br />
temperatures during the first hours from immersion.<br />
Experiments were realizes by Else [5] regarding<br />
the bacterial colonization of the hydrophile surface of<br />
metals (stainless steel, titanium and nickel) in<br />
variable conditions of temperature (30º C, 60º C and<br />
70º C) and humidity over a longer period of time<br />
(from a few days to 18 months). They observed an<br />
increase of adherent bacteria between 1.06∙10 2 cel/cm<br />
2 and 7.61 ∙10 2 cel/cm 2 at a temperature of 30º C on<br />
steel plates. They also observed a <strong>de</strong>crease of the<br />
number of bacteria from the first day for the plates<br />
exposed to high temperatures (60º C and 70º C),<br />
especially on those of nickel and steel.<br />
In what regards the role of the bacterial film in<br />
the mediation of invertebrate attachment and fouling<br />
formation, Lau et al. [35] realized experiments at<br />
different temperatures (16º C, 23º C and 30º C),<br />
noticing an increase in the number of bacteria from<br />
14.3∙10 3 cel/mm -2 at 16º C to 21.2∙10 3 cel/mm -2 at<br />
30º C. The experiments emphasized a more<br />
significant influence of the temperature on the<br />
biomass than on the bacterial <strong>de</strong>nsity.<br />
A number of experiments regarding the<br />
formation of biofilms in different conditions of<br />
temperature were realized by Di Bonaventura [36]<br />
together with other collaborators accomplish in 2007<br />
(4º C, 12º C, 22º C, 37º C) by Listeria<br />
monocytogenes on the hydrophile surface of glass,<br />
steel and the hydrophobe surface of polystyrene. The<br />
results emphasized a progressive increase on the<br />
surface at 4 ºC of 0.206, at 12 ºC BPI to 0.233 BPI,<br />
22º C to 0.366 BPI, in comparison to polystyrene and<br />
stainless steel. At 37º C the values are close to those<br />
from the three surfaces studied, but there is also<br />
greater species variability. Still, the most<br />
consi<strong>de</strong>rable growth of 1.275 was obtained on the<br />
hydrophobe surface of polystyrene.<br />
Bacterial <strong>de</strong>nsity registers an increase of the<br />
adherent bacteria with higher values for the biofilm<br />
formed in aquarium water kept at 18 ºC, compared to<br />
the one kept at 6 ºC. The values obtained are higher<br />
than those for seawater, which is due to the different<br />
physical and chemical properties of aquarium water<br />
and to the nutrients. Adherent marine bacteria attach<br />
151<br />
themselves to surfaces and form microcolonies in the<br />
first hour after immersion in the marine medium.<br />
They grow in size with the immersion period, data<br />
confirmed by [24].<br />
3.3 The formation of biofilms un<strong>de</strong>r the<br />
influence of salinity<br />
Variation of salinity was done in or<strong>de</strong>r to<br />
observe the influence of osmotic conditions on the<br />
process of bacterial adherence and the formation of<br />
the initial phases of biofilms. For the sli<strong>de</strong>s immersed<br />
in containers with seawater with 15g/l salinity, Figure<br />
3 displays an increase of bacterial <strong>de</strong>nsity to 12∙10 2<br />
cel/mm 2 one hour after immersion towards a<br />
doubling of this value to 25∙10 2 cel/mm 2 eight hours<br />
later.<br />
The sli<strong>de</strong>s were left over night for 12 hours and<br />
the following day there was a progressive increase of<br />
the cellular <strong>de</strong>nsity value of 49∙10 2 cel/mm 2 where<br />
there is a ten<strong>de</strong>ncy for a triple value towards 62∙10 2<br />
cel/mm 2 .<br />
In the case of containers with seawater with<br />
modified salinity (addition of osmosis water 10g/l),<br />
the bacterial <strong>de</strong>nsity increased to 4∙10 2 cel/mm 2 one<br />
hour after immersion to a double value of 8∙10 2<br />
cel/mm 2 after four hours and the progressive increase<br />
from 18 ∙10 2 cel/mm 2 after eight hours when there is<br />
a ten<strong>de</strong>ncy to triple the value of bacterial <strong>de</strong>nsity.<br />
After the 12 hour interval when the sli<strong>de</strong>s were left<br />
over night in containers, there is an increase of the<br />
cellular <strong>de</strong>nsity from 41∙10 2 cel/mm 2 24 hours after<br />
immersion to 54∙10 2 cel/mm 2 36 hours after<br />
immersion. The growth progression during the first<br />
12 hours in the case of the biofilms formed at a<br />
salinity of 15g/l is higher, with a value of 2.3 and<br />
displays a <strong>de</strong>crease after 24 hours to 1.1. In the case<br />
of the biofilms formed at a salinity of 10g/l, the<br />
growth progression is 2.4 during the first 12 hours<br />
and it <strong>de</strong>creases after 24 hours to 1.1. The difference<br />
between the two progressions is 2∙10 2 cel/mm 2 in the<br />
first 12 hours, it increases to 8∙10 2 cel/mm 2 24 hours<br />
after immersion and remains constant at this value<br />
until 36 hours.<br />
In the case of containers with seawater with<br />
modified salinity (addition of osmosis water 5g/l),<br />
Figure 4 displays an increase of bacterial <strong>de</strong>nsity<br />
from 2∙10 2 cel/mm 2 one hour after immersion to a
The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
double value of 4∙10 2 cel/mm 2 three hours later and<br />
the progressive increase to 9 ∙10 2 cel/mm 2 six hours<br />
later when the ten<strong>de</strong>ncy is for a triple value of the<br />
bacterial <strong>de</strong>nsity. The sli<strong>de</strong>s collected after 12 hours<br />
display a progressive increase of cellular <strong>de</strong>nsity from<br />
31∙10 2 cel/mm 2 24 hours after immersion to 49∙10 2<br />
cel/mm 2 36 hours later.<br />
The increase is accomplished based on a<br />
progression with a value of 2.3 in the first 12 hours in<br />
the case of the biofilms formed at 15g/l salinity and a<br />
<strong>de</strong>crease of this value to 1.1 after 24 hours.<br />
For the biofilms formed at 5g/l salinity, the<br />
value of the growth progression is 1.5 in the firs 12<br />
hours, which drops to 1.4 in the following 24 hours.<br />
Between the two growth progressions there are<br />
differences between the values of cellular <strong>de</strong>nsity.<br />
Thus, after 12 hours, the difference is 13∙10 2 cel/mm<br />
2 and it drops after 24 hours to 8∙10 2 cel/mm 2 , but<br />
increases after 36 hours to 13∙10 2 cel/mm 2 .<br />
While studying the colonization of surfaces by<br />
the bacterium B<strong>de</strong>llovibrio bacteriovorus, [32]<br />
realized experiments un<strong>de</strong>r the influence of different<br />
temperatures and observed the existence of a<br />
colonization ten<strong>de</strong>ncy and biofilm formation between<br />
3.4 g/l and 35 g/l. Salinity influenced the formation of<br />
biofilms even at values below 5 g/l, the number of<br />
adherent bacteria in the biofilm formed at 11g/l<br />
salinity being well over the expected one. At 4g/l<br />
salinity there is a <strong>de</strong>crease in the number of cells from<br />
3.5∙10 6 CFU/cm 2 to 3.8∙10 4 CFU/cm 2 five days after<br />
immersion.<br />
Some experiments regarding the role of<br />
bacterial biofilm were accomplished by [35] in the<br />
mediation of invertebrate attachment and<br />
microfouling formation at different temperatures and<br />
salinity values of 20g/l-34g/l. There is bacterial<br />
increase from 12.8∙10 3 cel/mm -2 to 20g/l la 21.2∙10 3<br />
cel/mm -2 at 34 g/l. The experiments reve<strong>ale</strong>d no<br />
significant correlation between salinity and bacterial<br />
<strong>de</strong>nsity in regards to biomass.<br />
Some experiments were accomplished in<br />
regards to the role of salinity (between 12g/l and<br />
80g/l) in the surface corrosion [37] achieves some<br />
experiments using stainless steel as substrate. They<br />
reve<strong>ale</strong>d the existence of a drop of cellular <strong>de</strong>nsity<br />
with the increase of water salinity, noticing a<br />
corrosion maximum at 35 g/l between 1.7 ∙10 9<br />
CFU/cm 2 and 2.1∙10 CFU/cm 2 for the aerobe species<br />
152<br />
analyzed. The experimental data have increased<br />
values compared to those obtained by our<br />
experiments.<br />
The values of cellular <strong>de</strong>nsity emphasize an<br />
increase correlated with the modification of salinity<br />
value as a whole, salinity increase from 5g/l to 10g/l<br />
and to 15g/l, the normal average value for seawater.<br />
These data are confirmed by the specialty literature as<br />
long as the increase is recor<strong>de</strong>d between certain<br />
optimum salinity limits.<br />
The <strong>de</strong>nsity values obtained when salinity was<br />
modified to 5g/l are lower than those for salinity from<br />
10g/l and 15g/l, which <strong>de</strong>monstrates that a possible<br />
supply of fresh water in the natural environment may<br />
influence the formation manner of the biofilms.<br />
Microcolonies form from the very first hours<br />
after the immersion of the hydrophile surfaces in the<br />
case of salinity variation as well. These data are<br />
confirmed by [24] in the specialized literature in the<br />
case of experiments for the formation of biofilms in<br />
static conditions.<br />
4. Conclusions<br />
The environmental factor such as temperature<br />
and salinity seems to influence bacterial adherence.<br />
The formation of the initial layers of the<br />
biofilms and their temporal dynamics in “in vitro”<br />
conditions <strong>de</strong>termines a progressive increase of<br />
cellular <strong>de</strong>nsity and the formation of microcolonies<br />
from the first hour after immersion in liquid medium.<br />
The modification of temperature and salinity values<br />
<strong>de</strong>termined a <strong>de</strong>crease of the total number of adherent<br />
cells, compared to the normal one on the hydrophile<br />
surface of glass, by mechanism(s) which are un<strong>de</strong>r<br />
investigation.<br />
5. References<br />
[1] ZOBELL C.E., 1943 – The effect of solid<br />
surfaces upon bacterial, J. Bacteriol., 46 (1): 39–<br />
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K.J. 1978 – How Bacteria Stick, Scientific<br />
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Aurelia Manuela Moldoveanu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
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lucrarii parctice, Universitatii Bucuresti, 320 pp.<br />
[23] HOVANEC T. A. AND DELONG E. F., 1996-<br />
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Associated with Freshwater and Marine Aquaria,<br />
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[24] MERRITT J. H., KADOURI D. E., AND.<br />
O’TOOLE G. A 2005- Growing and Analyzing<br />
Static Biofilms, Current Protocols in<br />
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[25] KUMAN A. AND PRASAD R., 2006 - Biofilms<br />
Jk. Science., 8 (1): 14 -17.<br />
[26] RUBIO C., 2002 - Comprehension <strong>de</strong>s<br />
mecanismes d’adhesion <strong>de</strong>s biofilms en milieu<br />
marin en vue <strong>de</strong> la conception <strong>de</strong> nouveaux<br />
motens <strong>de</strong> prevention, These <strong>de</strong> dcotorat, Paris,.<br />
216 pp.<br />
[27] SONAK S,BHOSLE N., 1995- A simple method<br />
to assess bacterial attachment to surfaces,<br />
Biofouling, 9(1): 31-38<br />
[28] HULEA A 1969 - Ghid pentru labortoarele <strong>de</strong><br />
micologie <strong>şi</strong> bacteriologie. Ed. Agrosilvică,<br />
Bucureşti.<br />
[29] FRY, J.C., 1990 – Direct methods and biomass<br />
estimation. Meth. Microbiol, 22: 41-85.<br />
[30] COCIASU A., LAZAR L. AND VASILIU D.<br />
2008 –New Ten<strong>de</strong>ncy in nutrient evolution from<br />
romanian costal waters, Cercetarii marine<br />
INCDM, 38: 7-23.
The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
[31] ROGERS J., DOWSETT A. B., DENNIS P. J.,<br />
LEE J. V., AND KEEVIL C. W., 1994 -<br />
Influence of Temperature and Plumbing<br />
Material Selection on Biofilm Formation and<br />
Growth of Legionella pneumophila in a Mo<strong>de</strong>l<br />
Potable Water System Containing Complex<br />
Microbial Flora, Appl. Environ. Microbiol., 60<br />
(5): 1585-1592.<br />
[32] KELLEY J. I., TURNG B.F, WILLIAMS H. N.,<br />
AND BAER M. L., 1997 - Effects of<br />
Temperature, Salinity, and Substrate on the<br />
Colonization of Surfaces In Situ by Aquatic<br />
B<strong>de</strong>llovibrios, Appl. Environ. Microbiol, 63: 84-<br />
90.<br />
[33] DI BONAVENTURA G., STEPANOVIC S.,<br />
PICCIANI C., POMPILIO A., PICCOLOMINI<br />
R., 2007- Effect of Enviromental Factors on<br />
Biofilm Formation by Clinical<br />
Stenotrophomonas maltophilia isolates, Folia<br />
Microbiol., 52 (1): 86-90.<br />
[34] TOREN L.A.., LANDAU L. KUSHMARO A.,<br />
LOYA Y. AND ROSENBERG E., 1998 -<br />
Effect of Temperature on Adhesion of Vibrio<br />
Strain AK-1 to Oculina patagonica and on Coral<br />
Bleaching , Appl. Environ. Microbiol, 64 (4):<br />
1379–1384.<br />
[35] LAU S.C.K, THIYAGARAJAN V., CHEUNG<br />
S.C.K, QIAN P.Y., 2005 - Roles of bacterial<br />
community composition in biofilms as a<br />
mediator for larval settlement of three marine<br />
invertebrates, Aquat Microb. Ecol, 38: 41–51.<br />
[36] DI BONAVENTURA G., PICCOLOMINI R.,<br />
PALUDI D., D’ORIO V., VERGARA A.,<br />
CONTER M. AND IANIERI A., 2007-<br />
Influence of temperature on biofilm formation by<br />
Listeria monocytogenes on various food-contact<br />
surfaces: relationship with motility and cell<br />
surface hydrophobicity, J. Appl. Microbio., 104 :<br />
1552–1561.<br />
[37] FRANCA F.P., FERREIRA C.A.,<br />
LUTTERBACH M.T.S., 2000- Effect of<br />
different salinities of a dynamic water system on<br />
biofilm formation, J. of Industrial Micro. &<br />
Bioteh., 25: 45-48.<br />
154
Density (10<br />
2 cel/mm 2 )<br />
Density (10<br />
2 cel/mm 2 )<br />
The formation of bacterial biofilms... / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
Early succesion of biofilms in containers over a period of 12 hours<br />
T 0=0 hours<br />
Sli<strong>de</strong>s Sea Water( Temp.6ºC)<br />
Sli<strong>de</strong>s Sea Water( Temp.18ºC)<br />
y = 2.3022x + 6.7253<br />
R 2 = 0.9354<br />
Table 1. The values of the seawater chemical parameters (liquid culture medium)<br />
Chemical parameters Sea water (zona litorala) Sea water<br />
(Aquarium)<br />
salinity 15.10 g/L 25.10 g/L<br />
pH 8.12 unit. 6.56 unit.<br />
P-PO4 0.74 µmoli/dm 3 63.80 µmoli/dm 3<br />
N-NO2 0.42 µmoli/dm 3 12.51 µmoli/dm 3<br />
N-NH4 1.13 µmoli/dm 3 5.46 µmoli/dm 3<br />
N-NO3 3.14 µmoli/dm 3 30.25 µmoli/dm 3<br />
Si-SiO4 21.16 µmoli/dm 3 0.24 µmoli/dm 3<br />
y = 1.9066x + 1.7912<br />
R 2 = 0.9668<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
Time (hours)<br />
Density (10 2 cel/mm2 )<br />
155<br />
66<br />
61<br />
56<br />
51<br />
46<br />
41<br />
36<br />
Early succesion of biofilms in containers over a period of 12 hours<br />
T 0=24 hours<br />
Sli<strong>de</strong>s Sea Water ( Temp.6ºC)<br />
Sli<strong>de</strong>s Sea Water (Temp.18ºC)<br />
y = 1.25x + 43.107<br />
R 2 = 0.9895<br />
y = 1.1429x + 35.714<br />
R 2 = 0.9922<br />
0 2 4 6 8 10 12 14<br />
Time (hours)<br />
Fig.1. The formation of a biofilm un<strong>de</strong>r the influence of temperature in containers with littoral seawater<br />
Ealy succesion of biofilms in containers over a period of 12 hours<br />
T 0=0 hours<br />
y = 2.6429x + 1.9121<br />
R 2 y = 2.5165x + 10.363<br />
R<br />
= 0.9883<br />
2 45<br />
40<br />
35<br />
Sli<strong>de</strong>s Aquarium (Temp.6ºC)<br />
Sli<strong>de</strong>s Aquarium ( Temp.18ºC)<br />
30<br />
25<br />
20<br />
= 0.8844<br />
15<br />
10<br />
5<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
Time (hours)<br />
Density (10 2 cel/mm2 )<br />
65<br />
60<br />
55<br />
50<br />
45<br />
40<br />
Early succesion of biofilms in containers over a period of 12 hours<br />
T 0=24 hours<br />
Sli<strong>de</strong>s Aquarium (Temp.6ºC)<br />
Sli<strong>de</strong>s Aquarium (Temp.18ºC)<br />
y = 1.125x + 47.839<br />
R 2 = 0.9883<br />
y = 1.2321x + 39.661<br />
R 2 = 0.9919<br />
0 2 4 6 8 10 12 14<br />
Time (hours)<br />
Fig.2. The formation of a biofilm un<strong>de</strong>r the influence of temperature in the containers with aquarium seawater
Aurelia Manuela Moldoveanu, Ioan Ar<strong>de</strong>lean / Ovidius University Annals, Biology-Ecology Series 14: 147-156 (2010)<br />
Density (10<br />
2 cel/mm 2 )<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
Ealty succesion of biofilms in containers over a peiod of 12 hours<br />
T 0=0 hours<br />
Sli<strong>de</strong>s Sea Water(Sal.10g/l)<br />
Sli<strong>de</strong>s Sea Water ( Sal.15g/l)<br />
y = 2.3022x + 6.7253<br />
R 2 = 0.9354<br />
y = 2.4341x - 0.2198<br />
R 2 = 0.9793<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
Time(hours)<br />
156<br />
Density (10 2 cel/mm2 )<br />
65<br />
60<br />
55<br />
50<br />
45<br />
40<br />
Ealry succesion of biofilms in containers over a period of 12 hours<br />
T 0= 24 hours<br />
Sli<strong>de</strong>s Sea Water(Sal.10g/l)<br />
Sli<strong>de</strong>s Sea Water(Sal.15g/l)<br />
y = 1.125x + 47.839<br />
R 2 = 0.9883<br />
y = 1.1607x + 39.446<br />
R 2 = 0.9816<br />
0 2 4 6 8 10 12 14<br />
Time (hours)<br />
Fig.3. The formation of a biofilm un<strong>de</strong>r the influence of salinity <strong>de</strong>crease (from 15g/l to 5 g/l) in the containers with<br />
littoral seawater<br />
Density (10 2cel/mm2 )<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
Early succesion of biofilms in containers over a period of 12 hours<br />
T 0= 0 hours<br />
Sli<strong>de</strong>s Sea Water ( Sal. 5g/l)<br />
Sli<strong>de</strong>s Sea Water (Sal.15g/l)<br />
y = 2.3022x + 6.7253<br />
R 2 = 0.9354<br />
y = 1.5385x - 0.2308<br />
R 2 = 0.9926<br />
0<br />
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14<br />
Time(hours)<br />
Density (10 2 cel/mm2 )<br />
69<br />
64<br />
59<br />
54<br />
49<br />
44<br />
39<br />
34<br />
29<br />
Early succesion of biofilm in containers over a period of 12 hours<br />
T 0=24 hours<br />
Sli<strong>de</strong>s Sea Water ( Sal.5g/l)<br />
Sli<strong>de</strong>s Sea Water (Sal.15g/l)<br />
y = 1.125x + 47.839<br />
R 2 = 0.9883<br />
y = 1.4286x + 28.714<br />
R 2 = 0.9627<br />
0 2 4 6 8 10 12 14<br />
Time (hours)<br />
Fig.4. Biofilm formation un<strong>de</strong>r the influence of salinity <strong>de</strong>crease (from 15g/l to 10g/l) in containers with littoral<br />
seawater
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE CLINICAL UTILITY OF ADITIONAL METHODS IN EFFUSIONS<br />
EVALUATION<br />
Ana Maria CRETU * , Mariana ASCHIE ** , Diana BADIU ** , Natalia ROSOIU ***<br />
*Ovidius University of Constanţa, Natural Sciences Faculty, Department of Biology,<br />
Mamaia Avenue, No. 124, Constanţa, 900552, Romania, e-mail: cretu_anamaria@yahoo.com<br />
** Clinical Emergency Hospital of Constanta, Department of Pathology, Tomis Avenue, No. 145, 900591,<br />
Constanta<br />
***Ovidius University of Constanţa, Medicine Faculty,Mamaia Avenue, No. 124, Constanţa, 900527, Romania<br />
________________________________________________________________________________________<br />
Abstract: Cells from reactive or hyperplasic mesothelium shed from body cavity surface, in various biological<br />
conditions, may present a wi<strong>de</strong> range of <strong>de</strong>viation from normal cellular morphology, making it difficult, or even<br />
impossible, to distinguish them from malignant cells by mean of purely cytological criteria. This study was<br />
carried out with the aim to evaluate if macroscopic features and cytologic formula can be used as potential<br />
diagnostic tool for distinguishing between malignant cells from reactive mesothelial cells in peritoneal effusions.<br />
We have examined the peritoneal effusions collected from 81 available cases, with a histological diagnosis<br />
known, from routine morphologic features. The various macroscopic parameters that were registered by<br />
macroscopic analysis inclu<strong>de</strong>d the registration of color, transparency and fluidity of peritoneal effusions.<br />
Comparing the results, there wasn`t found any relationship between peritoneal fluid containing cancer cells and<br />
liquid color. Cell smear appearance had a various cells populations and the quantitative analysis of effusions was<br />
not enough useful in establishing the final diagnosis.<br />
Keywords: peritoneal effusions, macroscopy, cytology, malign, benign<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The cytological diagnoses of serous effusions<br />
are usually ma<strong>de</strong> by routine cytomorphology with<br />
certainty, allowing treatment <strong>de</strong>cisions. Various<br />
studies have shown a sensitivity of 57.3% and<br />
specificity of 89% by conventional cytology for the<br />
<strong>de</strong>tection of malignant cells in effusion samples [1].<br />
The conventional cytology rate for i<strong>de</strong>ntification<br />
of neoplastic cells in effusions is about 60%. The rate<br />
of diagnostically equivocal effusions in routine<br />
cytology is <strong>de</strong>pen<strong>de</strong>nt on the volume of effusion<br />
examined, type of preparation and staining,<br />
experience of the examiner, and application of<br />
ancillary methods [2]. Peritoneal effusions are a<br />
frequently encountered clinical manifestation of<br />
metastatic disease, with breast, ovarian, and lung<br />
carcinomas and malignant mesothelioma leading the<br />
list [3, 4].<br />
Neoplastic cells that disseminate into cavities<br />
containing effusions are highly metastatic and possess<br />
a strong autonomous proliferative drive while<br />
concurrently being stimulatory of exudative<br />
effusions. The diagnosis of a malignant effusion<br />
signifies disease progression and is associated with a<br />
worse prognosis regardless of the tumor site of origin.<br />
Furthermore, cancer cells of different origins differ<br />
consi<strong>de</strong>rably in their biology and have unique<br />
phenotypic and genotypic characteristics [5].<br />
Primary cytomorphologic criteria of malignancy<br />
inclu<strong>de</strong> cellular aggregates, pleomorphism (variable<br />
cellular appearance), anisocytosis (variation in cell<br />
size), anisokaryosis (variation in nuclear size),<br />
multinucleation, prominent to irregular nucleoli,<br />
increased nuclear to cytoplasmic ratio, monomorphic<br />
cellular appearance, and increased mitotic figures.<br />
Hyperplastic mesothelial cells also may exhibit<br />
anisocytosis, anisokaryosis, increased nuclear to<br />
cytoplasmic ratio, binucleate and multinucleate, and<br />
scattered mitoses. Any situation that results in fluid<br />
accumulation within the body cavities can induce<br />
mesothelial cell hyperplasia and exfoliation with an<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The clinical utility of aditional metho<strong>de</strong>s... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010)<br />
abnormal cellular morphology [2]. Therefore, the<br />
differentiation between mesothelial cell hyperplasia<br />
and mesothelioma may be difficult or impossible.<br />
The first report of an intraoperative examination<br />
of peritoneal cytology to <strong>de</strong>tect subclinical metastases<br />
was presented in 1971. Patients with normal<br />
peritoneal cytological specimens had better survival<br />
rates than patients with abnormal findings, but only<br />
one abnormal cytologic specimen was found in early<br />
stage disease. [6]. Factors such as in patient versus<br />
outpatient management and associated procedural<br />
discomfort are important in the <strong>de</strong>cision making<br />
process, and the patient should participate in these<br />
subjective consi<strong>de</strong>rations [7].<br />
In addition, the etiology of the primary<br />
complaint is frequently multifactorial. However,<br />
malignant effusions recur, and therefore repeated<br />
paracentesis, especially if the fluid rapidly<br />
reaccumulates, is usually not a good long-term<br />
solution unless the patient’s overall prognosis and<br />
current condition prohibits a more invasive option.<br />
It is difficult to compare results and <strong>de</strong>termine<br />
the true efficacy of different techniques and agents<br />
because endpoints and response criteria as well as the<br />
extent and method of follow-up vary. Therefore<br />
various techniques should be used to increase the<br />
diagnostic accuracy of malignancy in serous<br />
effusions.<br />
2. Material and Methods<br />
This study was based on evaluation of 81<br />
available cases, with a histological diagnosis known,<br />
carried out in Emergency Clinical Hospital of<br />
Constanta – Pathological Anatomy Department<br />
(SCJUC ) from octobre 2007 to January 2010.<br />
Follow-up data were obtained from the Tumor<br />
Registry at SCJUC. Clinical charts of all the patients<br />
whose peritoneal fluid samples were sent for<br />
cytological examination during the study period were<br />
retrieved for relevant information.<br />
The fluid for cytological analysis was collected<br />
during laparotomy from the abdominal cavity. If no<br />
fluid was present, the peritoneal cavity was lavaged<br />
with saline solution, and the fluid was then collected<br />
for analysis.<br />
Giemsa stained and Papanicolaou stained sli<strong>de</strong>s<br />
were prepared from sediment obtained by<br />
158<br />
centrifuging the peritoneal liquid samples at 1500<br />
rpm for 5 minutes, using Shandon Cytospin<br />
preparations. After the centrifugation, the stained is<br />
fixed using alcohol (95% ethyl alcohol) as the<br />
fixative. Effusion cytology was studied from 40<br />
peritoneal effusions associated with at least one<br />
malignancy and 41 effusions collected from pacients<br />
with hepatic cirrhosis.<br />
We have examined the peritoneal effusions<br />
from routine macroscopic and cytologic features.<br />
Determination (the qualitative method) of<br />
cellular <strong>de</strong>nsity, specific weight and protein content<br />
from peritoneal fluid was performed by the Riwalta<br />
reactions [61]. Riwalta reaction is the reaction<br />
performed for differential diagnosis of exudates from<br />
transudates, based on precipitation of fibrin<br />
(insoluble protein, the main component of blood clot,<br />
a result of thrombin action on fibrinogen in plasma<br />
soluble, synthesized by the liver) meeting usual in<br />
inflammatory exudates (transudates usual, do not<br />
contain this fibrin) [10]. The reaction is positive<br />
when dripping the liquid examined in the mixture is<br />
obtaining an op<strong>ale</strong>scent, as a cloud. For obtaining the<br />
liquid peritoneal cytology formula, 100 cellular<br />
elements were measured from each smear cellular,<br />
thus directly establishing a percentage value.<br />
3. Results and Discussions<br />
From all 81 cases who <strong>de</strong>veloped peritoneal<br />
fluid, 41 were benign cases (associated with liver<br />
cirrhosis), 4 cases were associated with hepatic<br />
carcinoma, 4 cases with lung carcinoma, 18 cases<br />
with ovarian carcinoma, 4 cases with breast<br />
carcinoma, 9 cases with gastrointestinal carcinoma<br />
tract and 2 cases with peritoneal mesothelioma (Table<br />
1). The studied cases were divi<strong>de</strong>d into two groups:<br />
the 41 benign cases were inclu<strong>de</strong>d in lot I and 40<br />
cases of peritoneal fluid associated with cancer were<br />
inclu<strong>de</strong>d in lot II.<br />
Table 1. Peritoneal fluid distribution according to<br />
primary disease and the number of cases<br />
Lots Primary disease No of<br />
cases<br />
Lot I Hepatic cirrhosis 41<br />
(no=41)
Lot II<br />
(no=40)<br />
Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010)<br />
Hepatic cancer 4<br />
Ovarian cancer 18<br />
Gastrointestinal cancer 9<br />
Breast cancer 4<br />
Pulmonary carcinoma 3<br />
Peritoneal mesothelioma 2<br />
In terms of etiology, the highest number of cases<br />
groupt in lot II were shown to have ovarian origin,<br />
represented by ovarian carcinoma (n = 18) (45%),<br />
followed by cases associated with gastrointestinal<br />
carcinoma (n = 9) (22.5%), liver and breast<br />
carcinoma (n = 4) (10%), carcinoma lung (n = 3)<br />
(7.5%) and malignant mesothelioma (n = 2) (5%)<br />
(Fig.1).<br />
The type of neoplasia associated with most<br />
cases with peritoneal effusions accumulation proved<br />
to be represented by ovarian carcinoma.<br />
Fig. 1. Percentage distribution of cases according to<br />
the origin of cancer associated<br />
Most patients in Group II (32.83%) were within<br />
the range of ages 61-70 years (40%), followed by the<br />
51-60 range (35%). Cases registered with ovarian<br />
carcinoma were inclu<strong>de</strong>d in most (61.11%) in the 51-<br />
60 age range, those registered with gastrointestinal<br />
carcinoma and the liver were contained mainly in the<br />
41-50 age range, breast cancers, malignant<br />
mesothelioma and lung were within the range 61-70.<br />
(Fig. 2).<br />
159<br />
Fig. 2. The peritoneal fluid on different intervals of<br />
age (HC hepatic cancer, OC ovarian cancer, GIC<br />
gastrointestinal cancer, BC breast cancer, PC lung<br />
cancer, MM malignant mesothelioma)<br />
According to cancer staging [8],<br />
- stage I: generally inclu<strong>de</strong> small tumors without<br />
invasion and who are perfectly curable in most cases<br />
the prognosis favorable<br />
- stage II and III inclu<strong>de</strong>s tumors with local<br />
invasion of surrounding tissues and lymph no<strong>de</strong>s,<br />
therapeutic approach and prognosis are different<br />
<strong>de</strong>pending on the time cells and organ of origin,<br />
- stage IV: at this stage are in general inoperable<br />
tumors, metastasis and recurrence and a reserved<br />
prognostic survival, only 7 cases (17.5%) (2 / 2, and<br />
peritoneal mesothelioma 100% 5 / 18, 27.77%<br />
ovarian carcinoma) were rated as stage III, the rest<br />
(82.5%) fits into state IV, which shows that, as the<br />
stage progresses neoplasia, this is more prev<strong>ale</strong>nt<br />
peritoneal fluid (Fig.3).<br />
Fig. 3. Percentage of cases according<br />
to stage neoplasia
The clinical utility of aditional metho<strong>de</strong>s... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010)<br />
From all patients with histopathologic and<br />
clinical data that indicate malignancy, a number of 5<br />
patients (2 / 2, 100% associated with malignant<br />
mesothelioma, 1/4, 25% associated with hepatic<br />
carcinoma, ¼, 25% associated with breast carcinoma,<br />
1/3, 33.33% associated with lung carcinoma) were<br />
<strong>de</strong>ceased before drawing to the final study (a period<br />
of approximately three years from the accumulation<br />
of fluid in the peritoneal cavity), in all cases, the<br />
peritoneal fluid cytology recor<strong>de</strong>d the presence of<br />
malignant cells (Fig. 4).<br />
Fig. 4. Percentage of prognostic survival of patients<br />
inclu<strong>de</strong>d in the study (OC- ovarian cancer, GIC -<br />
gastrointestinal cancer, HC - hepatic cancer, BC –<br />
breast cancer, PC - pulmonary cancer, MM -<br />
malignant mesothelioma)<br />
Thus, the percentage of peritoneal fluid<br />
accumulation in the abdominal cavity is directly<br />
proportional to the tumor stage and also with the<br />
diagnosis of malignant peritoneal effusions, meaning<br />
that the progression of cancer is associated with an<br />
unfavorable prognosis. None of the patients with<br />
ovarian or gastric carcinoma were associated with an<br />
unfavorable prognosis, which indicates that this<br />
patients are available for a longer treatment.<br />
Neoplasia stage, histological gra<strong>de</strong> of neoplasia,<br />
positive cytology of peritoneal fluid and patients age<br />
(61-70 years) were correlated statistically with the<br />
prognosis.<br />
The first step in the analysis of peritoneal fluid<br />
was represented by analysis of the macroscopic point<br />
of view of biological material (peritoneal effusions).<br />
160<br />
Thus, it was recor<strong>de</strong>d: the extracted amount, the<br />
product color, transparency and its consistency.<br />
After macroscopic analysis, the most liquids<br />
from the group I was found to shown yellow color<br />
(from very light yellow to orange - yellow), and most<br />
fluid were transparent. In stead, the peritoneal<br />
effusions from group II had a variable macroscopic<br />
appearance: a number of 29/40 (72.5%) were intense<br />
yellow colored and transparents, many of them<br />
(17/29, 42.5% of all liquids associated with a<br />
carcinoma) had tissue fragments occupying<br />
approximately 25-50% from all quantity effusions,<br />
suspen<strong>de</strong>d in liquid; 5 (12.5%) showed a yelloworange<br />
fluid and opaque, and a total of six (15%)<br />
were hemorrhagic (<strong>de</strong>ep red), fluid and opaque (Fig.<br />
5). Of these, 19 / 40 (47.5%) had fragments of tissue<br />
suspen<strong>de</strong>d in peritoneal effusions. These fragments<br />
were then inclu<strong>de</strong>d in paraffin, stained and examined<br />
microscopically.<br />
We can say that the macroscopic analysis of<br />
peritoneal fluid, associated with cases of cancer are<br />
different from those associated with cases of cirrhosis<br />
only by this tissue fragments foun<strong>de</strong>d suspen<strong>de</strong>d in<br />
the effusions, with a capacity of discrimination of<br />
47.5%.<br />
Fig. 5. Peritoneal fluid: yellow (a) and hemorrhagic<br />
The registration of the important differences<br />
existing between macroscopic peritoneal fluids is<br />
essential, representing the first way in effusions<br />
discrimination (in transudates and exudates), so that,<br />
the material subjected can provi<strong>de</strong> useful information<br />
for further evaluation of the cells from cytological<br />
smears. It is known that bleeding effusions (<strong>de</strong>ep red)<br />
are often caused by a cancer and that these liquids<br />
often contain cancer cells [9].
Ana Maria Creţu et al. / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010)<br />
However, comparing the results, after<br />
performing peritoneal fluid cytology, with those<br />
obtained by macroscopic evaluation, of the 40<br />
effusions associated with at least one malignancy,<br />
only 6 (15%) were foun<strong>de</strong>d to be red colored<br />
(hemorrhagic), emphasizing that it does not exist any<br />
relationship between peritoneal fluid containing<br />
cancer cells and fluid color.<br />
After conducting the Riwalta reactions [10], the<br />
81 peritoneal effusions were classified in: 29<br />
(35.80%) transudates peritoneal fluid (with low cell<br />
<strong>de</strong>nsity and low protein content, which is usually<br />
accumulated in benign conditions) and 43 (53.08%)<br />
exudate (effusions with high cell <strong>de</strong>nsity and high<br />
protein content, which is accumulated most in<br />
malignant conditions), and 9 (11.11%) mixed,<br />
intermediate peritoneal effusions. Thus, peritoneal<br />
fluids were classified into three groups: group I<br />
(transudates), group II (intermediate, mixed) and<br />
group III (exudates) (Table 2).<br />
Table 2. Distribution of cases after Riwalta reaction<br />
Lots Primary<br />
cancer<br />
Lot I<br />
(n=41)<br />
Lot II<br />
(n=40)<br />
Transudates<br />
(N=29)<br />
Mixed<br />
(N=9)<br />
Exudates<br />
(N=43)<br />
CB 21 3 17<br />
CH (4) 1 1 2<br />
CO (18) 3 0 15<br />
CGI (9) 1 2 6<br />
CM (4) 2 2 0<br />
CP (3) 1 1 1<br />
MP (2) 0 0 2<br />
Since only 26/40, 65% of peritoneal effusions<br />
associated with different type of cancer resulted to<br />
have characters of exudates, and only 21/41, 51.21%<br />
of effusions associated with liver cirrhosis were<br />
shown to be transudates, it follows that, by<br />
conducting the Riwalta reaction, it can <strong>de</strong>termine the<br />
benign or malignant nature of effusions in a<br />
proportion of 58.10% (Fig.6).<br />
Cell smear appearance had a various cells<br />
populations and the quantitative analysis of effusions<br />
was not enough useful in establishing the final<br />
diagnosis. There were observed 7 cell types present<br />
in variable number. Proliferative capacity of tumor<br />
cells - tumor aggressiveness - is an important<br />
161<br />
element in cytology grading of malignancy, and was<br />
quantified by mitosis counting.<br />
Fig 6. The benign or malignant nature of effusions<br />
after conducting the Riwalta reaction<br />
In smears classified as benign, isolated cells<br />
represented 90% of total cells, cell groups recovered<br />
to a rate of 10%. 5% were represented by free<br />
nucleus or cells with damaged cytoplasm.<br />
Mesothelial cells (33%) (33 cells of 100<br />
elements) and lymphocytes (30%) were the majority<br />
cell type in the group of benign peritoneal effusions,<br />
followed by macrophages (17%), polymorphonuclear<br />
leukocytes (PMN) (9%) and erythrocytes (7%).<br />
Average of total number of mitosis foun<strong>de</strong>d in<br />
studied smears was 3mitosis/smears (Table 3).<br />
Cellular composition of effusions foun<strong>de</strong>d to<br />
be suspicious for malignancy was similar with the one<br />
of benign peritoneal effusions: mesothelial cells<br />
(28%) (28 cells of 100 items) and lymphocytes (26%)<br />
were the majority cell type, followed by neutrophils<br />
(13%), atypical cells, suspicious for malignancy<br />
(9%), erythrocytes (9%) and macrophages (7%).<br />
Average of total number of mitosis foun<strong>de</strong>d in<br />
studied smears was 7 mitosis/smears (Table 3).<br />
In the group of patients with malignant cancer,<br />
mesothelial cells represented 29% and erythrocytes<br />
21%, followed by lymphocytes (18%),<br />
polymorphonuclear leukocytes (16%), macrophages<br />
(4%) and malignant cells (4%), average of total<br />
number of mitosis was 9 mitoses / cell smear (Figure<br />
7).
The clinical utility of aditional metho<strong>de</strong>s... / Ovidius University Annals, Biology-Ecology Series 14: 157-162 (2010)<br />
Tabel 3. The percentage of cellular elements (%)<br />
BE* AE* ME*<br />
Mesothelial<br />
cells<br />
33 28 29<br />
Atipical cells 0 9 0<br />
Malignant cells 0 0 3<br />
erythrocytes, 7 9 21<br />
lymphocytes 30 26 18<br />
PMN 9 13 16<br />
macrophages 17 7 4<br />
mitosis / cell<br />
smear<br />
2 7 9<br />
Average 12,25 12,375 12,5<br />
standard<br />
Deviation<br />
13,15566 9,738546 10,12776<br />
p(t
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
SPATIO-TEMPORAL DYNAMICS OF PHYTOPLANKTON COMPOSITION AND<br />
ABUNDANCE FROM THE ROMANIAN BLACK SEA COAST<br />
Laura BOICENCO<br />
National Institute for Marine Research and Development „Grigore Antipa”<br />
300, Mamaia Bd., Constanta, 900581, Romania, e-mail: boicenco@alpha.rmri.ro<br />
__________________________________________________________________________________________<br />
Abstract: Based on more than 2,000 samples collected during 1996-2007, the paper <strong>de</strong>als with the taxonomic<br />
and ecological composition, spatio-temporal <strong>de</strong>velopment of phytoplankton blooms from waters of up to 50 m<br />
<strong>de</strong>pths laying on the Romanian Black Sea. The author i<strong>de</strong>ntified 396 species, varieties and forms, and assessed a<br />
<strong>de</strong>nsity mean varying among 417 and 3,376∙10 3 cells∙l -1 . Bacillariophyta phylum, with a number of 157 taxa and<br />
a <strong>de</strong>nsity mean of minimum 186.4 (in 2000) and maximum 2,311.9∙10 3 cells∙l -1 (in 1997), was the most numerous<br />
(39.6% of the total); Dinoflagellata was the second dominant group, represented in the communities with 85 taxa<br />
(21.5%); <strong>de</strong>nsity means ranged from 9.2 (in 2003) and 225.6∙10 3 cells∙l -1 (in 1997). Groups Chlorophyta and<br />
Cyanobacteria represented only 19.4 and 12.9%, respectively, from the total number of species. Species showing<br />
huge <strong>de</strong>velopments in the reference period were: the diatoms Skeletonema costatum, Cerataulina pelagica,<br />
Nitzschia <strong>de</strong>licatissima, Chaetoceros socialis, Cyclotella caspia and dinoflagellates Prorocentrum minimum,<br />
Heterocapsa triquetra and Scrippsiella trochoi<strong>de</strong>a.<br />
Keywords: taxonomic composition, ecological composition, phytoplankton blooms<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The early 1990s seemed to be a new<br />
beginning for the Black Sea ecosystem. After the<br />
“Mnemiopsis era” <strong>de</strong>scribing the 8 th <strong>de</strong>ca<strong>de</strong>,<br />
superimposed on the 20 years-long “eutrophication<br />
era” started in the 1970s, signs of improvement of<br />
its ecological state occurred, evi<strong>de</strong>nced by a<br />
reduction of the Danube river nutrient input, a<br />
<strong>de</strong>crease in the frequency of hypoxia conditions, an<br />
increase in fod<strong>de</strong>r zooplankton biomass, and a<br />
drop in M. leidyi’s abundance. The recovery of the<br />
ecosystem was attributed partly to the collapsing<br />
economy and agricultural production, and to some<br />
protective measures taken to control anthropogenic<br />
pollution in all the coastal countries.<br />
Due to their short life cycles and quick<br />
response to changes in their environment, the<br />
phytoplankton was sensitive to these new shifts,<br />
displaying a ten<strong>de</strong>ncy to “normal” status before<br />
eutrophication: <strong>de</strong>creased amplitu<strong>de</strong> and frequency<br />
of blooms, and a qualitative and quantitative<br />
structure similar to the period 1960-1970 rather<br />
than 1980-1990.<br />
So, between 1991 and 1996, only six maximum<br />
<strong>de</strong>nsities, higher than 10 6 cells·l -1 , were registered,<br />
compared to 13 in the1980s; among them, only two<br />
species produced ample bloom events: Prorocentrum<br />
minimum (53.1 and 93.7·10 6 cells·l -1 in the summer of<br />
1991 and 1995) and Microcystis pulverea (60.0·10 6<br />
cells·l -1 in the spring of 1991) [1].<br />
The range of algal groups was different from that<br />
of 1970s and 1980s, but quite similar to that of 1960-<br />
1970 with a reduction of non-diatom bloom amplitu<strong>de</strong><br />
and increase of numerical <strong>de</strong>nsity and especially<br />
biomass of diatoms. The reduction of non-diatoms<br />
coinci<strong>de</strong>d with <strong>de</strong>crease of nutrient stocks, especially<br />
of phosphates, which reached concentrations of 2.55<br />
µM·l -1 in 1991-1996, 2.8 times lower than in 1986-<br />
1990 [1].<br />
During 1995-1996, the Black Sea ecosystem<br />
showed abrupt shifts in all trophic levels, from primary<br />
producers to apex predators. This arose as a<br />
manifestation of concurrent changes in its physical<br />
climate induced by intensive warming of surface<br />
waters, as well as abrupt increases in the mean sea<br />
level and annual mean fresh water flux [2].<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
The aim of the present paper is to evaluate<br />
the spatio-temporal dynamics of the main<br />
taxonomic groups, and also the main bloomforming<br />
species during 1996-2007.<br />
2. Material and Methods<br />
Biological material was collected during<br />
seasonal surveys carried out within the scientific<br />
NIMRD’s programs, on board RV/STEAUA DE<br />
MARE, in the Romanian coastal waters laying<br />
between 43 0 50’-45 0 05’N and 28 0 50’–30 0 00’E (Fig.<br />
1). 2,018 quantitative samples were collected from<br />
472 stations covering the whole Romanian littoral<br />
at standard <strong>de</strong>pths (0, 10, 20, 30, 40, 50 and 60m),<br />
from the following profiles: Sulina, Mila 9, Sf.<br />
Gheorghe, Zaton, Portiţa, Chituc, Constanta,<br />
Mangalia.<br />
Fig.1. Sampling network<br />
The sampling used a NISKIN bottle; the water<br />
is transferred in 500ml bottles and preserved with<br />
formal<strong>de</strong>hy<strong>de</strong> 4%. In laboratory, the samples were<br />
processed using the MOROZOVA-VODIANITSKAIA<br />
and BODEANU’s methods [3, 4]. After two weeks of<br />
sedimentation, the supernatant liquid is siphoned<br />
off up to about 100ml. The sample is put in a small<br />
jar for another 10 days sedimentation. Before<br />
164<br />
microscopic processing, the sample is again siphoned<br />
off up to 10ml and stirring. 0.1ml of sample is<br />
examined un<strong>de</strong>r a ZEISS inverted microscope; the cells<br />
are counted and i<strong>de</strong>ntified at species or genus and the<br />
numerical <strong>de</strong>nsity is obtained relating the number of<br />
cells to a volume of 1 litre.<br />
Table 1 presents the environmental background<br />
(inorganic nutrients) of the annual phytoplankton<br />
<strong>de</strong>velopments. Phosphates showed a sharp <strong>de</strong>crease<br />
after 1997 down to a level similar to that before<br />
eutrophication. The inorganic total nitrogen<br />
concentrations have steadily <strong>de</strong>pleted ever since the<br />
1980s down to a minimum 9.48µM in 1985 followed<br />
by a relative long period, when these nutrients<br />
presented non-uniform oscillations. Among 1995 and<br />
2005, the levels of total inorganic nitrogen<br />
homogenously <strong>de</strong>creased, the maximum limit being<br />
situated between 10 and 15µM. In recent years their<br />
concentrations have began to increase to more than<br />
20µM, a value similar to that from 1980.<br />
With the exception of Si/N ratio, the molar<br />
ratios are still far by from the normal values,<br />
indicating that trophic anions do still not have optimal<br />
values for the normal <strong>de</strong>velopment of marine<br />
phytoplankton, although they show a <strong>de</strong>creasing<br />
ten<strong>de</strong>ncy. During the last period, the N/P ratio<br />
increased, due to an excessive <strong>de</strong>crease of phosphates<br />
and slight increase of inorganic nitrogen [5].<br />
Table 1. Multiannual mean of surface nutrient<br />
concentrations in coastal waters off Constanta<br />
Period 1983-‘90 1991-‘00 2001-‘05<br />
N-NO3 (µM) 6.90 5.90 7.98<br />
N-NH4 (µM) 5.11 7.06 6.12<br />
P-PO4 (µM) 6.54 1.86 0.49<br />
SiO4 (µM) 11.0 12.6 13.7<br />
3. Results and Discussions<br />
During 1996-2007, 396 microalgae species,<br />
varieties and forms belonging to seven phyla were<br />
i<strong>de</strong>ntified in the Romanian Black Sea waters (Fig. 1),<br />
the minimum number of 140 being found in 1996 and<br />
the maximum one in 2004. The most important group<br />
is Bacillariophyta, with 157 species, representing<br />
39.6% of the total; the second place is occupied by<br />
dinoflagellates, with 85 species (21.5%), followed by
Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
chlorophytes – 77 species (19.4%) and<br />
cyanobacteries – 55 species (12.9%); the rest of<br />
phyla (Chrysophyta, Euglenophyta, Cryptophyta),<br />
with 12, 8 and 6 species, respectively, constitute<br />
together only 6.6% of the total (Fig. 2).<br />
Fig. 2. Taxonomic composition.<br />
Ecologically, the phytoplankton represents a<br />
combination of autochthonous species, comprising<br />
euryhaline marine and brackish water forms (218),<br />
and alochthonous forms, comprising fresh-brackish<br />
and fresh water forms (178), reflecting the mixed<br />
marine water masses and riverine fresh waters<br />
which characterise the hydrologic regime of the<br />
Romanian sector.<br />
Table 2. Structure by ecologic groups.<br />
Phylum MarineFreshbrackish brackish<br />
Bacillariophyta 112 45<br />
Dinoflagellata 83 2<br />
Chlorophyta 0 77<br />
Cyanobacteria 9 42<br />
Chrysophyta 8 4<br />
Euglenophyta 2 6<br />
Cryptophyta 4 2<br />
Total 218 178<br />
Diatoms<br />
During 1996-2007 the averaged data for the<br />
whole Romanian littoral waters suggest that the<br />
communities are dominated by diatoms, both in<br />
terms of numeric <strong>de</strong>nsity and biomass. The<br />
multiannual mean of 833.5·10 3 cells·l -1 is 12.2<br />
times higher than dinoflagellates (68.5·10 3<br />
cells·l -1 ). The highest diatoms mean <strong>de</strong>nsity -<br />
2,311.9·10 3 cells·l -1 , achieved in 1997 was 10.2<br />
times higher than that recor<strong>de</strong>d for dinoflagellates<br />
165<br />
in the same year. The diatoms produced their highest<br />
mean <strong>de</strong>nsities during summer; in the summer of 1997,<br />
they excee<strong>de</strong>d 6 million cells per litre. When we were<br />
able to collect samples in winter, e.g. in 1999, we<br />
found out that many diatoms – such as S. costatum, C.<br />
caspia, Ch. socialis, N. tenuirostris began to vegetate<br />
in winter. So, we <strong>de</strong>tected communities very well<br />
constituted, higher than 10 6 cells∙l -1 . In five out of 12<br />
springs investigated (1999, 2003, 2004, 2006 and<br />
2007), the diatom populations were most abundant;<br />
they progressively <strong>de</strong>creased toward summer and<br />
autumn.<br />
Table 3 shows the diatom species with the<br />
highest <strong>de</strong>nsities in the Romanian Black Sea waters,<br />
between 1991 and 2007.<br />
Table 3. The highest <strong>de</strong>nsities of diatoms<br />
(10 6 cells∙l -1 )<br />
1996- 2001-<br />
Species<br />
2000 2007<br />
Cyclotella caspia 10.5 78.6<br />
Skeletonema costatum 24.4 37.3<br />
Nitzschia tenuirostris 1.8 15.5<br />
Cerataulina pelagica 8.2 10.0<br />
Chaetoceros socialis 22.2 7.5<br />
Skeletonema subsalsum 4.4 3.9<br />
N. <strong>de</strong>licatissima 0.6 2.5<br />
Small-sized diatom, Skeletonema costatum is an<br />
omnipresent species in the communities i<strong>de</strong>ntified not<br />
only at the Romanian littoral but in whole Pontic<br />
basin, producing most of the bloom events. Its<br />
maximum level was registered in July 2002, off<br />
Constanta (37.3∙10 6 cells∙l -1 ). Its second outburst<br />
occurred in the shallow waters of Mamaia Bay (where<br />
the sampling is carried out almost weekly); starting in<br />
the second half of March 1998, the phytoplankton<br />
communities were more and more abundant, attaining<br />
the value of 24.3∙10 6 cells·l -1 on March, 31. We have<br />
to remark that the communities from Mamaia Bay<br />
were almost monospecific, being constituted up to<br />
99.8% only by Skeletonema.<br />
Eurythermal and euryhaline species,<br />
Skeletonema vegetates abundant starting from winter,<br />
not only in Mamaia Bay, where usually attain over<br />
5∙10 6 cells∙l -1 , in January-March, but also in <strong>de</strong>eper<br />
waters off Constanta, where its concentrations reached
Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
3.6∙10 6 cells∙l -1 in January 1999. In the spring of<br />
2006, again in Mamaia Bay, S. costatum produced<br />
two other bloom events: 15∙10 6 cells∙l -1 (April, 25)<br />
and 11∙10 6 cells∙l -1 (May, 4) (Fig. 3), when the<br />
temperatures oscillated from 9.8 to 13.3 0 C and the<br />
salinity <strong>de</strong>creased gradually from 16.53 to 12.64<br />
and 9.04 PSU.<br />
In waters un<strong>de</strong>r the direct influence of the<br />
Danube, Skeletonema often produced <strong>de</strong>nsities<br />
ranged from 1.4 to 6.1∙10 6 cells·l -1 , both in spring<br />
and summer. But in September 1999, its<br />
populations were even richer at all the stations of<br />
the profile: 6.0 (Sulina), 8.1 (Mila 9), 7.7<br />
(Sf.Gheorghe) and 6.3 ∙10 6 cells·l -1 (Portita).<br />
Fig. 3. Long-term evolution of S. costatum blooms.<br />
However, the last blooms produced by<br />
Skeletonema are much lower than those recor<strong>de</strong>d<br />
in the period of maximum eutrophication. A good<br />
indicator of hypereutrophic waters, S.costatum<br />
showed overwhelming populations after 1970; for<br />
instance, between 1983 and 1986, S. costatum<br />
bloomed up to its highest value of 141.4∙10 6 cells∙l -1<br />
in April 1983.<br />
But, the most significant bloom event<br />
registered during the whole study period was<br />
generated by another diatom, Cyclotella caspia in<br />
the shallow waters of Mamaia. On May 3, 2001, it<br />
reached the value of 78.6∙10 6 cells·l -1 , which is 3.2<br />
times higher than S. costatum’s peak; the event was<br />
amplified by the abundant population of<br />
Skeletonema, raising the total <strong>de</strong>nsity up to<br />
84.82∙10 6 cells∙l -1 .<br />
Cyclotella gave another two important<br />
outbursts, but they were 7.6 and 4.0 times lower<br />
than the preceding one: in June 1999 (10.4∙10 6<br />
cells∙l -1 ) at Mamaia, and June 2005 (19.7∙10 6<br />
cells∙l -1 ) at Constanta (Fig. 4). Rich populations<br />
were found also in the northern sector, but never as<br />
high as those found in the southern area; the richest<br />
166<br />
one was i<strong>de</strong>ntified in waters from Sf.Gheorghe site<br />
(6.4∙10 6 cells·l -1 ). Anyway, these highest values are far<br />
from the exceptional <strong>de</strong>velopment recor<strong>de</strong>d by<br />
Cyclotella in 1981 (300∙10 6 cells∙l -1 ) [6].<br />
Fig. 4. Long-term evolution of C. caspia blooms.<br />
Chaetoceros socialis is the third diatom with<br />
frequent occurrence and <strong>de</strong>nsities higher than 100∙10 3<br />
cells·l -1 , but only two blooms were higher than 10∙10 6<br />
cells·l -1 : in June 1997, in front of the Danube Delta<br />
(Mila 9) (15.9∙10 6 cells·l -1 ), and in May 2000, at<br />
Mamaia (22.2∙10 6 cells∙l -1 ) (Fig. 5).<br />
Ch. socialis is a new entry the list of bloomforming<br />
species. During the period 1971-1990, only<br />
Ch. similis f. solitarius <strong>de</strong>veloped large<br />
concentrations: 13.2∙10 6 cells∙l -1 (between 1970 and<br />
1980) and 21.5∙10 6 cells∙l -1 in May 1988.<br />
Fig. 5. Long-term evolution of Ch. socialis blooms.<br />
However, in 1956, 1957 and 1961, SKOLKA cited<br />
Ch. socialis among the species producing some<br />
abundances higher than 10 6 cells·l -1 (its peak of<br />
2.6∙10 6 cells∙l -1 was attained in June 1957); generally it<br />
accompanied other bloom-forming species such as<br />
S. costatum [7].<br />
During the study period, another diatom group,<br />
including Cerataulina pelagica, Nitzschia<br />
<strong>de</strong>licatissima and N. tenuirostris, periodically<br />
contributed to increase the total phytoplankton<br />
abundances, and C. pelagica had a <strong>de</strong>nsity range from<br />
3 to 10 million cells per liter. But only two of
Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
Nitzschia’ species (N. tenuirostris and N.<br />
<strong>de</strong>licatissima) had occurred and had low <strong>de</strong>nsities.<br />
Apart from the N. tenuirostris’ single bloom<br />
produced in July 2006 in Mamaia Bay (15.5∙10 6<br />
cells∙l -1 ), the two species had <strong>de</strong>nsities higher than<br />
10 6 cells∙l -1 only in five and six years, respectively.<br />
N. tenuirostris started to vegetate intensely after<br />
1981, and reached its amplest bloom in the summer<br />
of 1989 - 74.8·10 6 cells·l -1 [6].<br />
Dinoflagellates<br />
With a long-term mean of 68.5∙10 3 cells∙l -1 ,<br />
the dinoflagellates comprised small percentages of<br />
the total phytoplankton, with a maximum of 17%<br />
in 2007; the highest mean <strong>de</strong>nsity was almost<br />
225.6·10 3 cells·l -1 in 1997. However, during two<br />
springs (1998 and 2007) the populations of<br />
dinoflagellates were <strong>de</strong>nser, with a <strong>de</strong>nsity mean of<br />
455.9·10 3 cells·l -1 . Between 1996 and 2007 a few<br />
species had concentrations higher than 10 millions<br />
cells per liter (Table 4) in different areas and years.<br />
Table 4. The highest <strong>de</strong>nsities produced by<br />
dinoflagellates (10 6 cells∙l -1 )<br />
1996- 2001-<br />
Species<br />
2000 2007<br />
Scrippsiella trochoi<strong>de</strong>a 0.3 25.3<br />
Heterocapsa triquetra 13.6 16.0<br />
Gymnodinium cf. aureolum - 10.7<br />
Prorocentrum minimum 10.5 9.0<br />
Mass growth of the Prorocentrum minimum,<br />
causing the water to turn red, was recor<strong>de</strong>d for the<br />
first time in the summer of 1974 along the<br />
Romanian littoral; the phenomenon was repeated in<br />
summers 1975 and 1976. Prorocentrum was the<br />
first dinoflagellate species reacting to the sud<strong>de</strong>n<br />
<strong>de</strong>crease in salinity (monthly average reached 13<br />
PSU, at Constanta) and huge increase in the<br />
concentrations of phosphates and nitrates (18 and<br />
11 times respectively higher than the period 1959-<br />
1960). Presence of such extraordinary blooms had<br />
never been noticed before: 181.5 (1974), 78.7<br />
(1975) and 111.6∙10 6 cells∙l -1 (1976), in the<br />
southern coastal waters, from Navodari to<br />
Mangalia [8]. During the following <strong>de</strong>ca<strong>de</strong>s, when<br />
167<br />
eutrophication got stronger and stronger, up to a<br />
climax from 1981-1990, the species attained even<br />
more prodigious proliferations, up to the value of<br />
807.6∙10 6 cells∙l -1 in July 1987. In fact, no other<br />
species would ever attain such <strong>de</strong>nsities as the<br />
Prorocentrum between 1971 and 1990. In the<br />
following years, the amplitu<strong>de</strong> of Prorocentrum’s<br />
blooms <strong>de</strong>creased, but in July 1995 it reached a<br />
<strong>de</strong>nsity of 93.7∙10 6 cells∙l -1 (Fig. 5), 8.6 times lower<br />
than its overwhelming <strong>de</strong>nsity in July 1987 [1].<br />
Fig. 5. Long-term evolution of P.minimum blooms.<br />
Besi<strong>de</strong> the diatom Skeletonema, P.minimum is<br />
the second most common species in the whole Pontic<br />
basin giving some of the highest blooms, especially in<br />
the NW sector. In our study period, P. minimum<br />
continued to have massive <strong>de</strong>velopments, but much<br />
lower than the previous ones: in June 1999 –10.4∙10 6<br />
cells∙l -1 and July 2001 – 8.93∙10 6 cells∙l -1 , both of them<br />
in Mamaia Bay. Here, up to 2001, during warm<br />
months, the species’ populations frequently excee<strong>de</strong>d<br />
1 million cells per litre; then, the <strong>de</strong>nsities were lower<br />
and lower, sometimes disappearing from samples.<br />
Three other dinoflagellates reached<br />
concentrations higher than 10∙10 6 cells∙l -1 , namely<br />
Heterocapsa triquetra, Scrippsiella trochoi<strong>de</strong>a and<br />
Gymnodinium cf. aureolum (Table 2). After<br />
<strong>de</strong>velopments, reaching a few or ten thousands cells<br />
per litre in the 1970s, H. triquetra and S. trochoi<strong>de</strong>a<br />
came to the list of the bloom-forming species, the first<br />
with a value of 97.6 ∙10 6 cells∙l -1 in the period 1971-<br />
1980, and the second one with a value of 25.8 ∙10 6<br />
cells∙l -1 in the period 1981-1990. After a period (1991-<br />
1996) of insignificant concentrations (highest value of<br />
1.9 ∙10 6 cells∙l -1 ) [1], Heterocapsa again reached high<br />
concentrations: 13.6∙10 6 cells∙l -1 , in May 1998 (at Mila<br />
9) and 10.3∙10 6 cells∙l -1 , in April 2000 (in Mamaia<br />
Bay) (Fig. 6). All along Romanian littoral, but<br />
especially in the northern sector, Heterocapsa<br />
produced substantial <strong>de</strong>nsities, ranging from 2.0 to
Spatio-temporal dynamics of phytoplankton composition.../ Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
5.0∙10 6 cells∙l -1 . S. trochoi<strong>de</strong>a and Gymnodinium<br />
cf. aureolum had only one single large bloom 25.2<br />
(August 2001) and 10.1∙10 6 cells∙l -1 (April 2007 in<br />
Mamaia Bay), respectively.<br />
Fig. 6. Long-term evolution of H. triquetra<br />
blooms.<br />
Other Groups<br />
Representatives of other groups did not<br />
achieve significant <strong>de</strong>nsities, only sporadically did<br />
some species dominate the communities, and<br />
cyanobacteria were the most numerous comparing<br />
with chlorophytes and chrysophytes. The species<br />
Merismopedia, Microcystis, Gloeocapsa,<br />
Oscilatoria and Aphanizomenon were the<br />
commonest and most frequent cyanobacteries<br />
(Table 4).<br />
Table 4. The highest <strong>de</strong>nsities produced by other<br />
groups (10 6 cells∙l -1 )<br />
Species<br />
1996-<br />
2000<br />
2001-<br />
2007<br />
CYANOBACTERIA<br />
Microcystis orae 272.0<br />
Microcystis pulverea 1.0 26.7<br />
M. aeruginosa 1.5 15.0<br />
Phormidium sp. 27 1.1<br />
CHRYSOPHYTA<br />
Emiliania huxleyi 1.3 1.1<br />
EUGLENOPHYTA<br />
Eutreptia lanowii 2.4 7.4<br />
Three species of Microcystis genus (M.<br />
pulverea, M. aeruginosa and M. orae) produced<br />
maximum <strong>de</strong>nsities between 12.8 and 271.9∙10 6<br />
cells∙l -1 , especially during summer of 2001-2003.<br />
The intense <strong>de</strong>velopment of these three small-sized<br />
168<br />
species of cyanophytes took place un<strong>de</strong>r increased<br />
values of temperature, simultaneously with <strong>de</strong>creased<br />
values of salinity and concomitant with optimal<br />
concentrations of nutrients [8]. The euglenophyte<br />
Eutreptia lanowii had a constant frequency of<br />
occurrence throughout the analyzed period, with<br />
maximum <strong>de</strong>velopments in June 2007 of 7.4∙10 6<br />
cells∙l -1 , in Mamaia Bay, and 2.8∙10 6 cells∙l -1 July 2002<br />
off Constanta.<br />
4. Conclusions<br />
Despite of the mitigation in the pressure exerted<br />
by anthropogenic eutrophication (i.e. <strong>de</strong>pletion of the<br />
inorganic nutrient concentrations down pre 1970<br />
values) and Mnemiopsis’grazing, the signs of the<br />
ecosystem rehabilitation i<strong>de</strong>ntified at the<br />
phytoplankton level occurred after 1990, seem to be<br />
very fragile and labile. That means if the necessary<br />
conditions (sud<strong>de</strong>n salinity reduction, sud<strong>de</strong>n increase<br />
in water temperature, high concentrations of specific<br />
biogenic compounds) are fulfilled, many of<br />
phytoplankters can produce ample blooms.<br />
Some of the species producing frequent and<br />
overwhelming blooms in the previous <strong>de</strong>ca<strong>de</strong>s, carried<br />
on generating significant blooms also in our study<br />
period (i.e. S. costatum, P. minimum, C. caspia etc).<br />
Other species have newly entered the list of bloomforming<br />
species, especially small-sized cyanophyte –<br />
M. pulverea (occurred during the period 1991-1996),<br />
M. orae, M. aeruginosa, Synecocystis sp., Gloeocapsa<br />
crepidinium, but also some large-sized diatoms<br />
Navicula sp., Amphora sp., Tabellaria sp. (after<br />
1996); all of them are alochthonous fresh-brackish<br />
species, introduced into the sea mainly by the Danube<br />
River. M. orae gave a <strong>de</strong>nsity of 272∙10 6 cells∙l -1 in the<br />
summer of 2000, the highest <strong>de</strong>nsity occuring after<br />
1990.<br />
Many times in the past, some of the bloom<br />
events, especially these of huge concentrations, were<br />
followed by fish and invertebrate mass mortalities. We<br />
used to consi<strong>de</strong>r that the species blooming at the<br />
Romanian littoral were dangerous only due to the<br />
negative impact produced as consequence of oxygen<br />
<strong>de</strong>pletion, reaching the threshold for lethal limits for<br />
invertebrates and fish. Such case took place in 1999,<br />
after a relatively high (10.4∙10 6 cells∙l -1 ) but longlasting<br />
bloom (June-July-August) produced by
Laura Boicenco / Ovidius University Annals, Biology-Ecology Series 14: 163-169<br />
Cyclotella caspia, in Mamaia Bay. Huge quantities<br />
of adult gobies, sole, plaice and turbot juveniles<br />
were washed up on the beaches or caught in<br />
lethargic condition from the sea by fishermen.<br />
As a matter of fact, HALLEGRAFF (1995)<br />
consi<strong>de</strong>rs that species vegetating in <strong>de</strong>nsities over<br />
5∙10 6 cells∙l -1 are harmful, since phytoplankton<br />
hyperproduction leads to regular violations of the<br />
ecosystem carrying capacity and severe economic<br />
losses to aquaculture, fisheries and tourism<br />
operations [9].<br />
However, some of algal species wi<strong>de</strong>ly<br />
distributed at the Romanian coastal waters, such as<br />
Chaetoceros socialis, C. curvisetus, Dichtyocha<br />
speculum, Ceratium fusus, can seriously damage<br />
fish gills, either mechanically or through<br />
production of hemolytic substances. Other ones,<br />
such as P. minimum, Dinophysis acuta,<br />
D. acuminata, D, sacculus, D. rotundata,<br />
M. aeruginosa are consi<strong>de</strong>red potentially toxic<br />
species, having the capacity to produce potent<br />
toxins, like DSP (diarrheic shellfish poisoning),<br />
that through the food chain could cause a variety of<br />
gastrointestinal illness to humans [9].<br />
The relationship between anthropogenic<br />
activities and changes in phytoplankton<br />
composition and diversity is one of the main<br />
objectives proposed in Harmful Algal Blooms<br />
research. Long time series of phytoplankton<br />
community storage in the NIMRD data base should<br />
be reconsi<strong>de</strong>red related to HAB increase.<br />
5. References<br />
[1] BODEANU N., RUTA G., 1998 – Development<br />
of the planktonic algae in the Romanian<br />
Black Sea sector in 1981- 1996. In<br />
Harmful Algae, B. Reguera, J.Blanco,<br />
L.Fernan<strong>de</strong>z, T. Wyatt (ed.) Vigo, Spain,<br />
1997: 188-191.<br />
[2] OGUZ T., DIPPNER J.W., KAYMAZ Z.,<br />
2006 – Climatic regulation of the Black Sea<br />
hydro-meteorological and ecological<br />
properties at interannual-to-<strong>de</strong>cadal time<br />
sc<strong>ale</strong>. Journal of Marine Systems, 6: 235-<br />
254.<br />
169<br />
[3] BODEANU N., 1987/1988 - Structure and<br />
dynamics of unicellular algal flora in the<br />
Romanian littoral of the Black Sea. Cercetari<br />
Marine, 20–21: 19–250.<br />
[4] MOROZOVA-VODIANITKAIA N.V., 1954 -<br />
The Black Sea phytoplankton, Tr. Sevastopol.<br />
Biol., 8: 11-99 (in Russian).<br />
[5] BSC, 2008. State of the Environment of the<br />
Black Sea (2001-2006/7). Edited by Temel<br />
Oguz. Publications of the Commission on the<br />
Protection of the Black Sea Against Pollution<br />
(BSC) 2008-3, Istanbul, Turkey: 23-49.<br />
[6] SKOLKA H.V., 1967 – Consi<strong>de</strong>raţii asupra<br />
variaţiilor calitative <strong>şi</strong> cantitative <strong>ale</strong><br />
fitoplanctonului litoralului românesc al Mării<br />
Negre. Ecologie Marină, Vol. 2: 193-293.<br />
[7] BODEANU N., ROBAN A., USURELU M.,<br />
1981 – Elemente privind structura, dinamica <strong>şi</strong><br />
producţia fitoplanctonului <strong>de</strong> la litoralul<br />
românesc al Mării Negre în perioada 1972-<br />
1977). Producţia <strong>şi</strong> productivitatea<br />
ecosistemelor acvatice. N. Botnariuc ed., Ed.<br />
Acad. Rom., Bucureşti: 42-50.<br />
[8] BODEANU N., ANDREI C., BOICENCO L.,<br />
POPA L.,. SBURLEA A, 2004 – A new<br />
trend of the phytoplankton structure and<br />
dynamics in the Romanian marine waters.<br />
Cercetari Marine, 35: 77-86.<br />
[9] VELIKOVA V., MONCHEVA S.,. PETROVA<br />
D, 1999 – Phytoplankton dynamics and red<br />
ti<strong>de</strong>s (1987-1997) in the Bulgarian Black Sea.<br />
Wat. Sci. Tech., Vol. 39, No. 8: 27-36.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
ASPECTS REGARDING THE BIODIVERSITY OF<br />
THE AQUATIC AND SEMI AQUATIC HETEROPTERA IN THE LAKES<br />
SITUATED IN THE MIDDLE BASIN OF THE OLT RIVER<br />
Daniela Minodora ILIE<br />
“Lucian Blaga” University, School of Sciences, Departament of Ecology and Environmental Protection,<br />
5-7 Dr. I. Raţiu Street, 550012, Sibiu, Romania<br />
__________________________________________________________________________________________<br />
Abstract: The present work analyzes the bio diversity of the aquatic and semi aquatic heteroptera belonging to<br />
four habitats, respectively lakes situated within the middle basin of the Olt River. From the collected biological<br />
material, consisting of 724 samples there were i<strong>de</strong>ntified 20 species of aquatic and semi aquatic heteroptera. We<br />
want to mention the presence of the species Paracorixa concinna in the lake in Cincşor, here being the single and<br />
only one appearance of this species till now in the basin of the Olt River. The different conditions of the<br />
researched habitats are to be seen in the structure of the communities of aquatic and semi aquatic heteroptera.<br />
The similitu<strong>de</strong> among the established communities is a quite a reduced one.<br />
Keywords: aquatic and semi aquatic heteroptera fauna, communities analysis, the middle basin of the Olt<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The aquatic and semi aquatic heteroptera lives<br />
in a great variety of habitats, from temporary swamps<br />
to big lakes, from brooks to small and big rivers, from<br />
continental waters to the surface of the oceans. The<br />
aquatic and semi aquatic heteroptera are consumers<br />
of the 2 nd <strong>de</strong>gree (the food base consisting of both<br />
<strong>de</strong>ad and alive prey).<br />
The present work proposes to evaluate the bio<br />
diversity of the communities of aquatic and semi<br />
aquatic heteroptera from the researched lakes that are<br />
situated in the following units of relief: Perşani<br />
Mountains, Făgăraş Mountanns and Hârtibaciu<br />
Plateau. The lakes are presented as follows:<br />
SO1: Bottomless Lake (Mateiaş)<br />
The lake is situated in Perşani Mountains,<br />
having the following coordinates: 45 0 59’ 08’’ N, 25 0<br />
20’<br />
20’’ E, at an altitu<strong>de</strong> of 522m. It is to be found on<br />
the left slope of the Olt River, being placed in the<br />
storages of the terrace allowing in this way the supply<br />
of the lake from the phreatic water.<br />
The lake, having a surface of approximately<br />
870m 2<br />
is surroun<strong>de</strong>d by willows. Phragmites<br />
communis and Typha latifolia covers about 5% from<br />
the banks area. The vegetation above and un<strong>de</strong>r the<br />
water is about 55-60% of the surface of the lake.<br />
There are to be found Lemna minor, L. trisulca,<br />
Spyrogyra sp., and also Ceratophyllum <strong>de</strong>mersum,<br />
Myriophyllum spicatum (a little). It is interesting to<br />
be mentioned the appearance in this station of the<br />
species Sagittaria latifolia and Potamogeton lucens<br />
that are seldom met in the middle basin of the Olt.<br />
SO2: Bâlea Lake<br />
The geographic coordinates are: 45 0 36’ 10’’ N,<br />
24 0 36’ 49’’E, at an altitu<strong>de</strong> of 2036m.<br />
It is a typical glacier lake sheltered in the so<br />
called Bâlea bucket, nearby the separating limit<br />
between the glacial circle and the former glacial<br />
valley. There is a mixed supply, this being the spring<br />
of the river Bâlea.<br />
The lake has a surface of 4.6 ha and a maximum<br />
<strong>de</strong>pth of 11.35.<br />
S03: Cincşor<br />
The lake is situated in the Hârtibaciu Plateau,<br />
having the geographic coordinates as follows: 45 0 49’<br />
36’’ N, 24 0 49’55’’ E, at an altitu<strong>de</strong> of 422m. It is an<br />
abandoned mean<strong>de</strong>r of the Olt River, which when<br />
there are big flood keeps in touch with the actual<br />
course of the river, being situated in its major<br />
riverbed. The supply of the lake is both from<br />
un<strong>de</strong>rground as well as superficial.<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010)<br />
The banks of the river are covered with willows<br />
(Salix alba, S. triandra, S. fragilis), which make the<br />
banks more stable and also give shadow to the water.<br />
SO4: Netuş<br />
The Netuş Lake is situated in the Hârtibaciu<br />
Plateau having the coordinates as follows: 46 0 03’<br />
55’’ N, 24 0 47’ 55’’ E, at an altitu<strong>de</strong> of 484m.<br />
The lake was arranged by people, its purpose<br />
being to reduce the flood. It also has a fish breeding<br />
interest, being placed in the major riverbed of the<br />
Hârtibaciu River. The vegetation is un<strong>de</strong>veloped.<br />
2. Material and Methods<br />
The biologic material was gathered during<br />
September-October 2001, September 2002, August<br />
and September 2004, From three stations there were<br />
gathered two samples: in September and October<br />
2001 from SO!, in September 2002 and August 2004<br />
from SO2, in September and October 2001 from<br />
SO3. From the station SO4 there was done only one<br />
gathering (October 2004). For the i<strong>de</strong>ntification of<br />
the species we used the <strong>de</strong>termination key of the<br />
following authors: [1], [2], and [3].<br />
There was calculated the relative abundance of<br />
each and every species from the researched habitats,<br />
diversity in<strong>de</strong>xes ά - Marg<strong>ale</strong>f (for general aspects,<br />
such as the number of species and the number of<br />
individuals) and Lloyd-Ghelardi (for the evaluation of<br />
heterogeneity) – and the indicator of percentage<br />
similitu<strong>de</strong> Renkonen, in accordance with [4].<br />
3. Results and Discussions<br />
As a result of the gatherings done during the<br />
periods mentioned before we i<strong>de</strong>ntified a number of<br />
20 species, from which 13 species are aquatic<br />
heteroptera (Heteroptera: Nepomorpha) and 7 species<br />
are semi aquatic heteroptera (Heteroptera:<br />
Gerromorpha), belonging to 9 families, presented in a<br />
number of 724 samples (table 1).<br />
The Corixidae family is the best represented<br />
taking into account the number of species (8 species),<br />
but consi<strong>de</strong>ring the number of the gathered<br />
individuals the Naucoridae family is on the first place<br />
(202 samples). At Mateiaş (SO1) we i<strong>de</strong>ntified 17<br />
species representing 50% from the total number of<br />
species that were gathered in the middle basin of the<br />
172<br />
river Olt (Ilie, 2009). Here is the only station where<br />
appeared the species Ilyocoris cimicoi<strong>de</strong>s, its<br />
presence being linked to the un<strong>de</strong>r water vegetation.<br />
Other species of aquatic heteroptera (Sigara striata,<br />
Sigara iactans, Notonecta glauca, Plea minutissima)<br />
are also well represented from the same reason. On<br />
the other si<strong>de</strong>, the vegetation above the water is<br />
favorable for the semi aquatic species (Microvelia<br />
reticulata, Mesovelia furcata and Mesovelia<br />
vittigera).<br />
The community of the aquatic and semi aquatic<br />
heteroptera from Mateiaş is <strong>de</strong>fined by high values of<br />
the relative abundance of the species Ilyocoris<br />
cimicoi<strong>de</strong>s (A=30.31%), Microvelia reticulata<br />
(A=20.85%), Gerris argentatus (A=16.62%) and<br />
Sigara striata (A=12.54%) and values less than 10%<br />
for the other species. There is to be noticed an<br />
equilibrate structure of the heteroptera community as<br />
two species of aquatic heteroptera and respectively<br />
two species of semi aquatic heteroptera represents<br />
about 40% from the total of the community. On<br />
assembly the aquatic heteroptera represent 60% and<br />
the semi aquatic heteroptera about 40% from the<br />
heteroptera community in the lake in Mateiaş (in the<br />
terms of relative abundance). The species Notonecta<br />
glauca is represented by an average number of<br />
individuals, the dimensions of the population being<br />
<strong>de</strong>termined by the big size and the predator behavior,<br />
which is extremely active.<br />
At the Bâlea Lake (SO2) we i<strong>de</strong>ntified only two<br />
species of heteroptera although there was done the<br />
some number of gatherings, the habitat being of the<br />
same kind (natural lake) and the relief unit the same,<br />
namely mountain. This fact is a result of the great<br />
differences of altitu<strong>de</strong>, which implies climate<br />
differences (especially the temperature, on which<br />
<strong>de</strong>pends the existence and the <strong>de</strong>velopment of the<br />
insects) as well as the vegetation (this being mainly a<br />
shelter against the predators). There was also noticed<br />
the fact that the species that were present in the Bâlea<br />
Lake are to be found in the Mateiaş Lake, too.<br />
At Cincşor (SO3) there were i<strong>de</strong>ntified 10<br />
species of aquatic and semi aquatic heteroptera. The<br />
most of the species belong to Corixidae family (4<br />
species). The other families are represented by one or<br />
maximum two species.<br />
In the aquatic and semi aquatic heteroptera<br />
community of the Cincşor Lake, Micronecta scholtzi
Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010)<br />
registered by far the highest value of the relative Table 3. The values of the Renkonen in<strong>de</strong>x<br />
abundance (A=46.00%). Some authors consi<strong>de</strong>red<br />
important the fact that fish eat several species of<br />
heteroptera, especially Corixidae, reducing in this<br />
way their populations [5]. The populations of the<br />
species Micronecta scholtzi were noticed in the shore<br />
S01- S01- S01- S02- S02- S03-<br />
S02 S03 S04 S03 S04 S04<br />
R. 23.87 19.40 21.00 0.00 22.22 0.00<br />
The similitu<strong>de</strong> between the aquatic and semi<br />
aquatic heteroptera communities in the 4 habitats was<br />
area of the aquatic habitat, a shadowed area and<br />
without un<strong>de</strong>rwater plants, having a reduced <strong>de</strong>pth,<br />
which is not favorable for fish, this being the<br />
explanation of the abundance of this species in the<br />
heteroptera community. We also want to notice the<br />
presence of the species Paracorixa concinna, here<br />
being the only once it was registered till now in the<br />
basin of the Olt River [6].<br />
At Netuş (SO4) there were i<strong>de</strong>ntified two<br />
species of semi aquatic heteroptera: Gerris lacustris,<br />
being collected 7 individuals and Microvelia<br />
reticulata, 2 individuals being collected. In this case<br />
the number of species is a much reduced one because<br />
the ecologic conditions are not proper for these<br />
heteroptera. We want to notice that both species are<br />
semi aquatic ones, these being less sensitive than the<br />
aquatic species regarding the volume and the <strong>de</strong>nsity<br />
of the un<strong>de</strong>rwater as well as the floating vegetation.<br />
Table 2. The values of the diversity in<strong>de</strong>xes ά<br />
obtained for every collecting station<br />
In<strong>de</strong>x / Station S01 S02 S03 S04<br />
Marg<strong>ale</strong>f 2.46<br />
3<br />
Lloyd-Ghelardi 0.68<br />
6<br />
0.91<br />
0<br />
0.91<br />
8<br />
2.30<br />
1<br />
0.71<br />
3<br />
0.45<br />
5<br />
0.76<br />
4<br />
The values of Marg<strong>ale</strong>f in<strong>de</strong>x are quite high for<br />
the habitats SO1 (2.463) and SO3 (2.301)<br />
respectively low for the habitats SO2 (0.910) and<br />
SO4 (0.455) (table 2). The higher values of the in<strong>de</strong>x<br />
show that there were better conditions in the habitat<br />
for the heteroptera species.<br />
The Lloyd-Ghelardi in<strong>de</strong>x, varying between 0<br />
and 1, shows for the researched habitats a relatively<br />
homogenous repartition of the individuals on the<br />
species, representing around 70% of the optimum<br />
value. SO2 is an exception having a higher value<br />
because of the i<strong>de</strong>ntification of individuals number<br />
closed to the species number.<br />
173<br />
established having as a base the Renkonen in<strong>de</strong>x,<br />
calculated with data of relative abundance of the<br />
species. It came out that there was a quite low<br />
similitu<strong>de</strong> (table 3).<br />
4. Conclusions<br />
There were i<strong>de</strong>ntified 20 species, from which<br />
we noticed the species Paracorixa concinna, at<br />
Cincşor being the only registration in the basin of the<br />
Olt River.<br />
The number of the i<strong>de</strong>ntified species in every<br />
habitat differs quite a lot (among 2-17 species) as<br />
well as the abundances of different species within the<br />
communities that establish them in those habitats (for<br />
example in SO1, the only station where the species<br />
Ilyocoris cimicoi<strong>de</strong>s was present, this being also the<br />
most abundant; in SO3 Micronecta sholtzi registered<br />
by far the highest value of the relative abundance).<br />
These show the variety of the conditions that are<br />
existent in those lakes; for the aquatic and semi<br />
aquatic heteroptera the quality of the habitats is<br />
connected with the altitu<strong>de</strong>, damming, the<br />
<strong>de</strong>velopment of the aquatic vegetation, the fish<br />
population, etc. The similitu<strong>de</strong> between the<br />
communities of aquatic and semi aquatic heteroptera<br />
established in those 4 habitats is quite a low one.<br />
5. References<br />
[1] DAVIDEANU, ANA, 1999. Contribuţii la studiul<br />
heteropterelor acvatice din România, Teza <strong>de</strong><br />
doctorat, Univ. ”Al. I. Cuza”, Ia<strong>şi</strong>, 427 pp.<br />
[2] JANSSON, A., 1986. The Corixidae (Heteroptera)<br />
of Europe and some adjacent regions, Acta Entom.<br />
Fennica, 47: 1-92.<br />
[3] POISSON, R., 1957. Hétéroptères aquatiques<br />
(Faune <strong>de</strong> France), 61: 1-263.<br />
[4] SÎRBU, I., BENEDEK ANA MARIA, 2004.<br />
Ecologie practică, Univ. Lucian Blaga, Sibiu, 1-<br />
264.
Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010)<br />
[5] PAPÁČEK, M., 2001. Small aquatic and ripicolous<br />
bugs (Heteroptera: Nepomorpha) as predators and<br />
prey, Eur. J. Entomol., 98: 1-12.<br />
[6] ILIE, DANIELA MINODORA, 2009.<br />
Heteropterele acvatice <strong>şi</strong> semiacvatice din<br />
bazinul mijlociu al Oltului, Ed. Altip, Alba-Iulia,<br />
1-279.<br />
174
Daniela Minodora Ilie / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010)<br />
Table 1. The i<strong>de</strong>ntified species of aquatic and semi aquatic heteroptera from the researched habitats and the<br />
values of the relative abundance<br />
Gathering Station S01 S02 S03 S04<br />
Taxon<br />
Individuals<br />
number<br />
A% Individuals<br />
number<br />
174<br />
A% Individuals<br />
number<br />
A% Individuals<br />
number<br />
Fam. Gerridae<br />
Gerris argentatus 110 16,62 5 10,00<br />
Gerris<br />
odontogaster 3 0,45<br />
Gerris lacustris 1 0,15 7 77,78<br />
Fam. Veliidae<br />
Microvelia<br />
reticulata 138 20,85 1 33,33 2 22,22<br />
Fam.<br />
Hydrometridae<br />
Hydrometra<br />
stagnorum 1 0,15 11 22,00<br />
Fam<br />
Mesoveliidae<br />
Mesovelia furcata 21 3,17 4 8,00<br />
Mesovelia vitigera 10 1,51 1 2,00<br />
Fam. Corixidae<br />
Micronecta<br />
(Dichaetonecta)<br />
scholtzi 23 46,00<br />
Corixa punctata 6 0,91<br />
Hesperocorixa<br />
linnaei 2 0,30<br />
Paracorixa<br />
concinna 1 2,00<br />
Sigara<br />
(Retrocorixa)<br />
limitata 1 0,15<br />
Sigara (Sigara)<br />
striata 83 12,54<br />
Sigara (Subsigara)<br />
iactans 45 6,80 1 2,00<br />
Sigara<br />
(Vermicorixa)<br />
lateralis 1 2,00<br />
Fam. Naucoridae<br />
Ilyocoris<br />
cimicoi<strong>de</strong>s 202 30,51<br />
Fam. Nepidae<br />
Nepa cinerea 2 0,30 1 2,00<br />
Fam.<br />
Notonectidae<br />
A%
Aspects regarding the biodiversity... / Ovidius University Annals, Biology-Ecology Series 14: 171-175 (2010)<br />
Notonecta viridis 2 0,30<br />
Notonecta glauca<br />
Fam. Pleidae<br />
20 3,02 2 66,67<br />
Plea minutissima<br />
Individuals<br />
15 2,27 2 4,00<br />
number per<br />
662 3 50 9<br />
gathering station<br />
Species number<br />
per gathering<br />
station<br />
17 2 10 2<br />
175
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
PROGRAM OF PREVENTION AND CONTROL OF FUNGUS INFESTATION OF<br />
GRAIN AND FODDER , HUMAN AND ANIMAL PROTECTION AGAINST<br />
MYCOTOXINS<br />
drd.ing. Ioan Aurel POP*, conf. dr. Augustin CURTICĂPEAN**, drd.ing. Alin GULEA*, dr. Cornel PODAR*,<br />
ing. Iustina LOBONTIU*.<br />
* Staţiunea <strong>de</strong> Cercetare Dezvoltare penru Creşterea Bovinelor Mureş,<br />
str. Principală 1227, Sângeorgiu <strong>de</strong> Mureş, jud Mureş.<br />
** Universitatea <strong>de</strong> Medicină <strong>şi</strong> Farmacie Târgu Mureş<br />
__________________________________________________________________________________________<br />
Abstract: mycotoxins contained in forages may yield to cause different health issues on farm livestock as<br />
<strong>de</strong>creasing the forage intake and bioconversion, serious illness and <strong>de</strong>ath. Food and Agriculture Organization<br />
(FAO) appreciates on global level that 25% of agricultural products are contaminated with mycotoxins. These<br />
compounds contaminate feeds before and after harvesting. Food quality monitoring on each stage, especially due<br />
to it’s fungal potential risk is very important for the <strong>de</strong>velopment of antifungal strategies adapted to local<br />
conditions. Thus, through a research project witch involves the quantification of mycotoxins concentrations from<br />
feed and food samples taken from different farms located in Central Region of Transylvania we managed to<br />
<strong>de</strong>velop a new method of <strong>de</strong>tection and quantification of three mycotoxins. The paper work presents a part of<br />
activities performed in a research project and comprises their results on preventing and control of funguses and<br />
mycotoxins.<br />
Keywords: mycotoxins, fungus, crops, methods.<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
Food safety has become one of the directions<br />
very important area to protect and improve the<br />
quality of life. To ensure all elements contributing to<br />
the increase of consumer protection and food quality,<br />
<strong>de</strong>velop new methods of control, as simple, low<br />
resource consuming, while used in normal conditions<br />
[1]. Thus, eliminating sources of toxic advanced<br />
occurring in food composition is a major goal. One<br />
such source is the species of fungi producing<br />
mycotoxins, which are found in most foods of plant<br />
origin whose storage / storage is ina<strong>de</strong>quate, but<br />
worse is that we find and their metabolites in animal<br />
products, products from infested feeding.<br />
Monitoring primary storage conditions, and<br />
assessment on a representative sample of infestation<br />
by specific analysis will recommend specific methods<br />
of prevention / treatment of <strong>de</strong>veloping adverse<br />
effects of mycotoxins in crops.<br />
In a research project has <strong>de</strong>veloped a new<br />
method for <strong>de</strong>tection and quantification of three<br />
mycotoxins for monitoring the infection status of feed<br />
and food grain with mycotoxins in various units and<br />
areas located in Region Development Centru. [2]<br />
The paper also presents results of experiments:<br />
- Study the behavior of wheat, barley, tritic<strong>ale</strong>s<br />
and corn hybrids tested in comparative culture from<br />
the years 2008, 2009 from SCDCB Mures and their<br />
hierarchy according to their resistance to disease<br />
attack;<br />
- Testing of eight plant protection products for<br />
disease prevention and control in cereals in climatic<br />
conditions in 2009 and monitoring the behavior of<br />
fungici<strong>de</strong>s in the production.<br />
2. Material and Methods<br />
Thurough the research project "Complex<br />
program of prevention and control of fungus<br />
infestation for grain and fod<strong>de</strong>r for providing animal<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010)<br />
wealth and consumer protection’ has achieved a<br />
status of monitoring infestation of feed and food<br />
grain with mycotoxins in various units and sectors<br />
located in the Development Region Center. Action<br />
was initiated in early June in a maximum period of<br />
susceptibility to the inci<strong>de</strong>nce of mycotoxins <strong>de</strong>posit<br />
being ma<strong>de</strong> by the team of researchers from SCDCB<br />
Mures Tg Mures and SC AGROFITOPLANT<br />
PharmacyLtd.<br />
Sampling activity was ma<strong>de</strong> taking account of<br />
Regulation (EC) NO. 401/2006 laying down the<br />
procedures for sampling and analysis methods for<br />
official control of mycotoxins in food.<br />
In total 180 samples were taken from 44 production<br />
units, which have in operation a total: 14 450 ha of<br />
arable land, 8960 cattle, 29,170 porcine, 36,640<br />
birds, 8792 sheep. Average area of farms covered<br />
operating is 328 hectares.<br />
Of the total samples: 145 samples were<br />
concentrated (maize grain, flour, PVM's, milk<br />
pow<strong>de</strong>r) and 35 samples of forage (silage, half hay,<br />
hay, grains, beet chips, etc.). [1]<br />
The quantification of mycotoxins in the<br />
samples, at the UMF Mures (Mures University of<br />
Medicine and Pharmacy) has <strong>de</strong>veloped a new<br />
method to quantify the simultaneous separation of<br />
mycotoxins by liquid chromatography HPLC using a<br />
DIONEX Ultimate 3000 with UV <strong>de</strong>tection<br />
simultaneously on different channels. Optimization<br />
method was performed to <strong>de</strong>termine simultaneously,<br />
using an ordinary system, more relevant mycotoxins<br />
present in samples of feed corn stored for eight<br />
months.<br />
Based on production and behavior have<br />
established multi fenophasic comparative cultural<br />
components subject to this project, DC M01 and M02<br />
with wheat varieties, tritic<strong>ale</strong> and barley. (Table 1)<br />
Table 1. Crop ranking regarding yield DON and<br />
ZON.<br />
Specia/Soiu<br />
l<br />
Producti<br />
a medie<br />
(Kg/ha) Isc<br />
Ierarh<br />
i<br />
zare<br />
I GRAU 6920<br />
Ariesan(Mt 6780,9 120,20 V<br />
7243,5 161,01 I<br />
178<br />
II<br />
Ar<strong>de</strong>al 1<br />
Magistral 7159,1 130,59 III<br />
Renan 6536,0 114,56 VI<br />
Exotic 7047,9 133,56 II<br />
Gasparom 7070,5 122,90 IV<br />
Turda<br />
14/98 6671,0 105,98 VIII<br />
Apullum 6851,8 110,33 VII<br />
TRITICAL<br />
E 7699<br />
Plai(Mt) 7798,7 120,20 III<br />
Titan 7971,4 123,85 II<br />
Trilstar 7501,8 104,89 V<br />
Stil 7590,3 117,51 IV<br />
00474T1-1 7632,1 125,30 I<br />
II ORZ 6481<br />
Gerlac(Mt) 6570,8 110,00 I<br />
Regal 6683,2 97,12 III<br />
Plaisant 6189,1 98,81 II<br />
For testing resistance to major pests and<br />
diseases of maize hybrids grown in the area was<br />
established in late April (2008.2009) a crop of corn<br />
hybrids compared with 24 (S = 1000 m) in the<br />
experimental field of the resort located in Sg Mures.<br />
The main observations ma<strong>de</strong>: plant vigor, flowering<br />
time, date of silk, drought resistance,<br />
Helminthosporium sp., Puccini sp., Ustillago sp.<br />
Attack of Fusarium sp., The number of sterile plants,<br />
the number of broken and fallen plants, resistance to<br />
attack pest and grain production.<br />
The content <strong>de</strong>termination of mycotoxins was<br />
performed at UMF Targu Mures (University of<br />
Medicine and Pharmacy. Mures) SPC Mures (Mures<br />
Public Health Center) Promovert laboratories in<br />
Champagne, France (company Bayer). [1]<br />
3. Results and Discussions<br />
Precision method for <strong>de</strong>termining meets the<br />
minimum relative standard <strong>de</strong>viation (with values in a<br />
field of ± 15%) for quality control samples measured
Ioan Aurel Pop et al. / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010)<br />
in both samples the same day and comparisons<br />
between samples from different days.<br />
Minimum limits of quantification for the three<br />
analytes / mycotoxins (2.88 ng / mL AflaB1, 2.88 ng<br />
/ mL respectively OchrA 14.4 ng / mL Zeara) met the<br />
requirements of precision and accuracy so that the<br />
relative standard <strong>de</strong>viation to be inclu<strong>de</strong>d in a field ±<br />
20% for both measurements on the same day as well<br />
as those performed on different days and the<br />
difference between mean calculated and nominal<br />
values (BIAS%) is also contained in a maximum field<br />
of ± 20%.<br />
Records on the evi<strong>de</strong>nce provi<strong>de</strong>d by corn and<br />
also on samples from various forage plants show an<br />
infestation of their importance to all three classes of<br />
mycotoxins, so Aflatoxin B1 and Ochratoxin A with<br />
zear<strong>ale</strong>none. Infestation levels are relatively high,<br />
regulated levels overruns are much more frequent and<br />
more significant if the first two Mycotoxins -<br />
Aflatoxin B1 respectively Ochratoxin A. Thus, the<br />
calculated values for concentrations of mycotoxin<br />
present in almost all unknown samples analyzed<br />
exceed permissible concentrations and regulated at<br />
European level. [2]<br />
High levels of mycotoxins found in animal feed<br />
and is probably due to the chosen period when this<br />
work started in early June, during which cereal stocks<br />
are running, close grain with a one year old storage<br />
warehouses before the process is Cleaning for storing<br />
grain harvest.<br />
Using production data obtained, the<br />
observations on different varieties fenophase attack<br />
on disease resistance, <strong>de</strong>oxyniv<strong>ale</strong>nol and<br />
zear<strong>ale</strong>none content in samples taken at harvest 16<br />
varieties of cereals were ranked using a synthetic<br />
in<strong>de</strong>x calculated Isc this.<br />
Results show that there are differences between<br />
varieties in terms of mycotoxins but not the values<br />
obtained exceeding the maximum allowed by law<br />
(1250 ng / g DON, and 100 ng / g Zon).<br />
Maize, based on production, moisture at<br />
harvest, percentage of plants broken and fallen and<br />
observations of vegetation during the attack on<br />
disease resistance of a synthetic in<strong>de</strong>x was calculated<br />
to ease the process generally ranking of cultivars.<br />
Results of tests carried out in laboratories SC<br />
Bayer SRL Promovert in Champagne, France<br />
reinforce the lessons learned so far. Of the seven<br />
179<br />
variants examined only version control - untreated<br />
with fungici<strong>de</strong> containing <strong>de</strong>oxyniv<strong>ale</strong>nol was above<br />
the limit of quantitation of 220 ng / g, respectively<br />
440 ng / g value in joining the legal permissible limit<br />
of 1250 ng / g DON. [1]<br />
% spice sanatoase<br />
101<br />
100<br />
99<br />
98<br />
97<br />
96<br />
95<br />
94<br />
93<br />
92<br />
95<br />
Martor<br />
netratat<br />
100 100<br />
Folicur Solo<br />
250 EW<br />
97<br />
100<br />
Tilt 250 EW Duett Ultra<br />
Produsul<br />
Prosaro 250<br />
EC<br />
97 97<br />
Nativo 300<br />
SC<br />
Falcon 460<br />
EC<br />
Fig. 1. Fungici<strong>de</strong>s effect in Fusarium removal<br />
from Ariesat wheat variety at Tg Mures.<br />
4. Conclusions<br />
Interpreted data show that the current<br />
methodology for preparing samples for analysis /<br />
quantification of mycotoxin content of substances of<br />
category has limits too generous. Thus, extraction of<br />
these substances (of which there are complex<br />
matrices) respecting the standardized methods, shows<br />
a lower sensitivity, which leads to highlighting of<br />
quantities / concentrations lower than actual. .[2]<br />
The existence of evi<strong>de</strong>nce over the maximum<br />
levels allowable by law certify the importance of this<br />
research and the need for a regional research<br />
antimycotic.<br />
Climatic conditions of the agricultural year 2008<br />
- 2009, characterized by high temperatures<br />
throughout the crop growing season and low rainfall<br />
than -114 mm limited attack foliar and ear diseases,<br />
and the effect of crop protection products was not<br />
very visible. For further research would require more<br />
years of study to catch different climates.<br />
Large assortment of hybrid corn study allows<br />
farmers to select hybrids with high production<br />
potential and adaptability to the conditions of the<br />
area. To limit the attack of diseases and in particular<br />
Fusarium in seed must be transmitted primarily by<br />
limiting attack Pyrausta which facilitates infection
Program of prevention and control of fungus… / Ovidius University Annals, Biology-Ecology Series 14: 177-180 (2010)<br />
how damaging fungal diseases of plants and causes<br />
breaking of preventing <strong>de</strong>ployment of mechanized<br />
harvesting in good condition. [3]<br />
5. References<br />
[1] POP I., GULEA A., CURTICĂPEAN A.,<br />
PODAR C. , 2009 - Program complex <strong>de</strong><br />
prevenire <strong>şi</strong> combatere a infestării cu miceţi la<br />
cere<strong>ale</strong> <strong>şi</strong> plante furajere pentru asigurarea<br />
bunăstǎrii anim<strong>ale</strong>lor <strong>şi</strong> protecţia<br />
consumatorilor, Raport <strong>de</strong> progres Transa a II-a.<br />
[2] A. CURTICĂPEAN, FELICIA TOMA,<br />
MONICA TARCEA, MANUELA<br />
CURTICĂPEAN, VICTOR SĂMĂRGHITAN,<br />
I. POP, A. GULEA, 2009 - Optimizarea unei<br />
meto<strong>de</strong> HPLC <strong>de</strong> separare <strong>şi</strong> <strong>de</strong>terminarea<br />
simultană a unor micotoxine din porumb,- Noi<br />
tendinţe <strong>şi</strong> strategii in chimia materi<strong>ale</strong>lor<br />
avansate. Institutul <strong>de</strong> Chimie Timişoara,<br />
Timişoara.<br />
[3] Pop I., Gulea A., Curticăpean A., Podar Cornel,<br />
2009 - ‚Program complex <strong>de</strong> prevenire <strong>şi</strong><br />
combatere a infestării cu miceţi la cere<strong>ale</strong> <strong>şi</strong><br />
plante furajere pentru asigurarea bunăstǎrii<br />
anim<strong>ale</strong>lor <strong>şi</strong> protecţia consumatorilor’- Raport<br />
<strong>de</strong> progres Transa I.<br />
180
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
DATA ON THE DYNAMICS OF SOME MICROBIAL GROUPS IN SOILS<br />
WITH DIFFERENT TROPHIC STATUS IN CUMPĂNA REGION (DOBROUDJA)<br />
Elena DELCĂ<br />
Ovidius University of Constanţa, Faculty of Natural Sciences and Agricultural Sciences<br />
Mamaia Avenue, No. 124, Constanţa, 900527, Romania,<br />
Doctoral school Biology, Specialization Ecology<br />
__________________________________________________________________________________________<br />
Abstract: The aim of paper was to assess the effect of administration of organic amendments on the dynamics of<br />
the abundance of microbial groups significant in nutrient cycling in soils. Abundance of total culturable bacteria<br />
ranged from 19.93x10 6 UFC/g dry soil, to 501.79x10 6 UFC/g dry soil. When soil was supplemented with manure<br />
microbial <strong>de</strong>nsity showed a significant increase 501.79x10 6 UFC/g dry soil compared with control variant.<br />
Bacterial <strong>de</strong>nsity increased significantly as value, too, following the administration of specific biofertilizers<br />
(Biovin, Bactofil Professional; Mycos Green), up to 142.13 x106UFC/g dry soil. Inorganic fertilizers did not<br />
have a positive effect on microbial <strong>de</strong>nsity values, being more or less similar to those reported for the control.<br />
Our preliminary data show that organic amendments with complex composition have a direct effect on the<br />
abundance and diversity of soil and influence indirectly the microbial metabolism and nutrient cycling rate.<br />
Keywords: humus, microorganisms, bioactivators, fertility<br />
_________________________________________________________________________________________<br />
1. Introduction<br />
To start and propose a suitable biological soil<br />
reconstruction plan it was necessary to initiate a<br />
series of observations and experiments in a<br />
characteristic agroecosystem of Dobroudja (Cumpana<br />
commune) in or<strong>de</strong>r to assess the current biological<br />
status. Using new agricultural technology, and adding<br />
different fertilizers the experiments have the aim to<br />
improve the number and activity of soil<br />
microorganism and indirectly to enhance the rate of<br />
organic matter <strong>de</strong>composition. This would improve<br />
over time the soil structure and restore the stock of<br />
humus in the soil.<br />
2. Material and Methods<br />
The experiments have taken place on a 7.5 ha<br />
plot situated in the outsi<strong>de</strong> of Cumpana, in Constanta<br />
district. Josef wheat was cultivated on the entire area,<br />
which was divi<strong>de</strong>d in 7 variants, each variant being<br />
administered a different type of fertilizer in different<br />
quantities and periods, as follows:<br />
- Variant I - only chemical fertilizers - 100kg/ha<br />
N15P25K15 in autumn, 150kg/ha NH4NO3 at the<br />
beginning of spring;<br />
- Variant II – Biovin organic fertilizer 400kg/ha and<br />
Biovin 30 of l/ha, ½ at herbici<strong>de</strong> stage and ½ at flour<br />
stage;<br />
- Variant III – gar<strong>de</strong>n soil - 15t/ha in autumn;<br />
- Variant VI – l/ha of Biovin 30, ½ at herbici<strong>de</strong> stage<br />
and ½ at flour stage;<br />
- Variant V – Biovin 150kg/ha administered during<br />
sowing, 150kg/ha NH4NO3, 40kg/ha at the beginning<br />
of spring, 50kg/ha at herbici<strong>de</strong> stage and 60kg/ha at<br />
flour stage;<br />
- Variant VI – Biovin 375kg/ha, liquid Biovin 30 of<br />
l/ha, ½ at herbici<strong>de</strong> stage and ½ at flour stage, 1mc<br />
Green Mycos, 1l Bactofil Professional;<br />
- Variant – March – were not applied amendments.<br />
Biovin Fertilizers are being administered for the<br />
first time in Dobrogea.<br />
Biovin is being produced through a<br />
technological process from grape kernels. 12 years of<br />
western research proved the following: it aerates the<br />
soil, improves it (it contains up to 70% humus<br />
makers), and purveys all plants with nutritive<br />
elements and biostimulators, it enriches the soil with<br />
ISSN-1453-1267 © 2010 Ovidius University Press
Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010)<br />
microorganisms that create humus, it strengthens the<br />
roots and it multiplies the percentage of smooth roots<br />
and radicular wintergr;<br />
Bactofil Professional is a product for<br />
improving the soil biological quality and contains<br />
nitrogen fixing bacteria phosphate-solubilization<br />
bacteria, and heterotrophic bacteria that stimulates<br />
the <strong>de</strong>composition of organic matter.<br />
Green Mycos is a product containing<br />
arbuscular mycorrhizal fungi and a number of factors<br />
that stimulate the establishment of symbiosis,<br />
improving the soil quality up to 20 years. [1]<br />
Experiments began in autumn 2009 by sampling<br />
the soil at a <strong>de</strong>pth of 15 cm approximately, followed<br />
by quantification of some agrochemical (humus,<br />
indice N2) and biological parameters (bacteria, free<br />
N2-fixing bacateria, actinomycetes, microfungi).<br />
Quantitative <strong>de</strong>termination of microbial<br />
abundance was done by <strong>de</strong>cimal dilutions of soil<br />
followed by inoculation of known quantities on solid<br />
nutrient media. For this purpose, after weighing the<br />
samples were inoculated on culture medium with a<br />
specific composition. Thus, to <strong>de</strong>termine the number<br />
of total culturable heterotrophic bacteria it has been<br />
used nutrient:<br />
- agar medium [2], [3] [4] - (pulvis yeast extract<br />
2.5 g, peptone 0.2 g, Agar 17-20g. It was sterilized<br />
20 min at 120 o C);<br />
- free N2-fixing bacteria on Ashby medium, [5]<br />
(15g Mannitol; g K2HPO4 0.2, MgSO4 ∙ 7H2O 0.5 g,<br />
0.2 g NaCl, CaSO4 ∙ 7H2O 0.1 g, CaCO3 5g, Agar<br />
17-20g. was sterilized 30 min at 115 o C).<br />
Determination was ma<strong>de</strong> on the environment<br />
actinomicete Czapeck – Dox ( 3g NaNO3; 1g<br />
K2HPO4; 0,5g MgSO4 ; 0,5g KCl; FeSO4 traces;<br />
Sucrose 30g; 17-20g Agar; pH 5,5; it was sterilized<br />
30 min at 115 o C) [3], [4], [7] and the abundance of<br />
microfungi was <strong>de</strong>termined on Sabouraud medium<br />
(CaCl2 0.5g, 0.1g K2HPO4, KH2PO4 0.1g, 10%<br />
MoO3 0.1ml, 0.05ml FeCl3 10%, was sterilized 30<br />
min at 115 o C).<br />
The total number of bacteria per gram of soil<br />
was calculated using the formula: no. bacteria,<br />
actinomycetes, microfungi = X colonies x dilution x<br />
10 x 100/100-U where X = average of colonies<br />
grown on culture medium, 10 = balancing coefficient<br />
of 0.1 ml of inoculum in the reporting of dilution soil<br />
U% = soil moisture. [8]<br />
182<br />
3. Results and Discussions<br />
The initial estimations have reve<strong>ale</strong>d a relatively<br />
low abundance variability between different<br />
experimental variants.<br />
Thus, the lowest abundance was <strong>de</strong>tected in<br />
variant VI, heterotrophic bacteria having a mean<br />
abundance of 19.93 x 10 6 CFU/g dry soil (Fig. 1).<br />
The abundance was highest instead on variant<br />
II, in which case the total number of heterotrophic<br />
bacteria reached 45.45 x 10 6 CFU / g dry soil (Fig.<br />
1).<br />
Fig. 1 Bacterial <strong>de</strong>nsity distribution in the initial<br />
stage of the experiment (October 2009)<br />
Changes in microbial abundance in<br />
experimental and control reflect the heterogeneity of<br />
normal physicochemical and trophic conditions of the<br />
soil, the values recor<strong>de</strong>d can be consi<strong>de</strong>red normal<br />
for chernozem soil type.<br />
Fig. 2 Distribution of heterotrophic bacterial <strong>de</strong>nsity<br />
after six months of application of amendments (May<br />
2010)
Elena Delca / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010)<br />
After six months of application of organic and<br />
inorganic amendments microbial abundance showed<br />
consi<strong>de</strong>rable changes in some cases, so the highest<br />
<strong>de</strong>nsity was recor<strong>de</strong>d in the experimental group<br />
fertilized with manure, variant III, in which case we<br />
<strong>de</strong>termined a <strong>de</strong>nsity of 501.79 x 10 6 CFU/g dry soil<br />
(Fig. 2). The number of bacteria also increased<br />
significantly in variant VI up to 142.13 x 10 6 CFU/g<br />
dry soil (Fig. 2).<br />
Paradoxically, after six months I have noticed<br />
<strong>de</strong>crease of heterotrophic bacteria in variant I, which<br />
might be explained by the effect of administration of<br />
chemical fertilizers and organic substance<br />
consumption by bacteria. In autumn 2009 the initial<br />
amount of organic matter in the form of crop residue<br />
remaining after harvest <strong>de</strong>creased gradually as the<br />
<strong>de</strong>composition and microbial consumption<br />
progressed and provi<strong>de</strong> sufficient nutrients to<br />
maintain viable bacterial population as numerous as<br />
in the beginning of the experiment. In other variants<br />
(II, IV and V) microbial <strong>de</strong>nsity presented weakly<br />
peaks compared with the control, and ranged between<br />
30.73 x 10 6 CFU/g soil dry and 46.31 x 10 6 CFU/g<br />
soil dry (Fig. 2), situation observed also in control<br />
variant.<br />
At the beginning of the experiment, <strong>de</strong>nsity of<br />
free nitrogen-fixing bacteria was relatively low, (Fig.<br />
3), ranging from 10.3 x 10 5 CFU/g dry soil to 26.7 x<br />
10 5 CFU/g dry soil. In case of variants I, III, V and<br />
VI values are very close to those recor<strong>de</strong>d for control<br />
(Fig. 3).<br />
Fig. 3 Changes of abundance of nitrogen-fixing<br />
bacteria (October 2009)<br />
183<br />
The highest number recor<strong>de</strong>d for variants II and<br />
IV ranging between 22.7 x 10 5 CFU/g dry soil and<br />
26.7 x 10 5 CFU/g dry soil, rely on local trophic<br />
conditions.<br />
In any case, there was a certain uniformity of<br />
abundance of nitrogen-fixing bacteria beginning of<br />
the experiment. At this stage relatively low<br />
abundance of nitrogen-fixing bacteria could be due to<br />
higher quantity of organic substance that stimulates<br />
competition within heterotrophic bacterial<br />
populations.<br />
Fig. 4 Change in binding <strong>de</strong>nsity micro N2, after six<br />
months (May 2010)<br />
After six months of adding the fertilizers our<br />
estimations reve<strong>ale</strong>d a significant increase of the<br />
number of nitrogen-fixing bacteria, including that of<br />
the control (Fig. 4).<br />
Most evi<strong>de</strong>nt increase of abundance of this<br />
group occurred I in variant III, where we recor<strong>de</strong>d<br />
about 195.81 x 10 5 CFU/g dry soil (Fig. 4).<br />
Significant increases were recor<strong>de</strong>d in the case<br />
of variants II, IV and VI, where abundance ranged<br />
from 90.44 x 10 5 CFU/g dry soil to 127.39 x 10 5<br />
CFU/g dry soil (Fig. 4).<br />
In variant I and V, values were close to the<br />
abundance <strong>de</strong>termined for control (Fig. 4).<br />
Table 1. Agrochemical analysis conducted in autumn<br />
2009<br />
Nr.<br />
Crt.<br />
Variant % mg/Kg<br />
Humus Indice N2<br />
1 V1 2.9 0.14
Data on the dinamics of some microbial groups... / Ovidius University Annals, Biology-Ecology Series 14: 181-184 (2010)<br />
2 V2 2.95 0.14<br />
3 V3 3.21 0.15<br />
4 V4 3.07 0.15<br />
5 V5 2.83 0.13<br />
6 V6 3.09 0.15<br />
7 M1 3.16 0.15<br />
Table 2. Agrochemical analysis conducted after 6<br />
months (May 2010)<br />
Nr.<br />
Crt.<br />
Variant % mg/Kg<br />
Humus Indice N2<br />
1 V1 3.07 0.15<br />
2 V2 3.09 0.15<br />
3 V3 3.36 0.17<br />
4 V4 3.12 0.15<br />
5 V5 3.07 0.15<br />
6 V6 3.12 0.15<br />
7 M1 3.24 0.16<br />
Further information relative <strong>de</strong>termination of<br />
humus were not noted substantial increases for the<br />
period un<strong>de</strong>r review (Table 1, Table 2), which is<br />
un<strong>de</strong>rstandable due to the short observation time<br />
insufficient to i<strong>de</strong>ntify significant changes in the<br />
humus content.<br />
4. Conclusions<br />
Dynamics of the total number of heterotrophic<br />
bacteria presented significant changes after<br />
application of amendments. The most significant<br />
increase occurred in the variant enriched with<br />
manure, trend was also observed in the case of<br />
variant VI in which soil was treated with Biovin.<br />
The total number of nitrogen fixing bacteria<br />
showed a spectacular increase after six months of<br />
amendments application, effect that can be attributed<br />
only in part as a result of fertilizer.<br />
Results of soil chemical and microbiological<br />
analysis reveal a low contribution of microorganisms<br />
to the improvement of the soil fertility and microbial<br />
biodiversity. To improve the biological quality of soil<br />
it is necessary to increase the biomass of<br />
microorganisms in the soil by adding bacteria, and<br />
184<br />
their activities by introducing large amounts of<br />
organic matter.<br />
5. References<br />
[1] BERCA M, 2008 – Probleme <strong>de</strong> ecologia solului.<br />
Editura ceres, 2008: 43-63.<br />
[2] BERGEY’S, 1986 - Manual of Sistematic<br />
Bacteriology, vol. 2, Williams and Wilkins,<br />
Baltimore, USA, 4087: 1075-1079<br />
[3] CLARK F, 1965 - Agar plate method for total<br />
microbial count. Method for Soil Analysis, vol.2:<br />
1460-1465 Amercian Society for Agronomy,<br />
Madison, WL.<br />
[4] FLORENZANO G, 1983 - Fondamenti di<br />
microbiologia <strong>de</strong>l rerreno, Reda Ed, Firenze, 630:<br />
115-136.<br />
[5] PAPACOSTEA P, 1976 - Biologia solului, Ed.<br />
Ştiinţifică <strong>şi</strong> Enciclopedica, Bucureşti, 272: 81-<br />
259.<br />
[6] PITT JL, 1991 - A Laboratory Gui<strong>de</strong> to common<br />
Penicillium Species, USA, 184: 129-135.<br />
[7] TSUNEO WATANABE, 2001 - Pictorial Atlas of<br />
Soil and Seed Fungi, Morphologies of Cultured<br />
Fungi and Key to Species – Second edition, CRC<br />
Press, 504: 230-236.<br />
[8] DUMITRU M, TOTI M, VOICULESCU A-R,<br />
2005 – Decontaminarea solurilor poluate cu<br />
compu<strong>şi</strong> organici, Ed. Sitech, Bucureşti, 364:<br />
262-266.
Ovidius University Annals of Natural Sciences, Biology – Ecology Series Volume 14, 2010<br />
THE AGRICULTURAL POTENTIAL OF PHOSPHOGYPSUM WASTE PILES<br />
Lucian MATEI<br />
Pescarusului Street, Bl CP1, Sc B, Ap 18, Navodari, Constanţa County, Romania,<br />
e-mail: mat_lucian@yahoo.com<br />
__________________________________________________________________________________________<br />
Abstract: The cultivation of Salix sp. on the phosphogypsum waste piles started from the wish to discover a<br />
cheap, efficient, and ecological covering method. For this purpose, Salix alba and Salix fragilis cuttings were<br />
used, as they were collected from an area adjacent to the town of Navodari. Some Salix fragilis cuttings were<br />
collected from the trees that grew spontaneously on the waste piles. The species Salix alba is newly introduced in<br />
the ecosystem of the phosphogypsum waste pile. The species of the genus Salix are dioicous. As they are not<br />
fertile, S. alba and S. fragilis are often crossbred in nature, creating hybrids, the most popular being S. x rubens.<br />
The large number of hybrids of the genus Salix offers them increased capacity to adapt and exist in the most<br />
various environmental conditions. The purpose of the project is to i<strong>de</strong>ntify a species or a hybrid that, given the<br />
life conditions on the phosphogypsum waste pile, should offer a consi<strong>de</strong>rable quantity of wood mass per ha in<br />
or<strong>de</strong>r to collect and exploit it as solid fuel.<br />
Keywords: Salix, phosphogypsum waste piles, ecological reconstruction, phytoreparation<br />
__________________________________________________________________________________________<br />
1. Introduction<br />
The i<strong>de</strong>a of cultivating Salix sp. on the<br />
phosphogypsum waste piles started from the wish to<br />
discover a cheap, efficient, and ecological covering<br />
method.<br />
The phosphogypsum waste pile number 3, which<br />
belongs to the S.C. Fertilchim – Marway S.A.<br />
company, is the result of massive accumulations of<br />
phosphogypsum obtained by the wet method of<br />
making phosphorus fertilizers. The waste pile is<br />
rectangular and has a surface of approximately 21 ha.<br />
In 1996, it was removed from the technological flux<br />
and a poor vegetation settled spontaneously on its<br />
surface over the next few years. A study regarding<br />
flora, accomplished in 2009, i<strong>de</strong>ntified 35 species of<br />
plants [1]. The dominant species is Puccinellia<br />
distans, a grass that prefers salty soils (Poaceae) [2].<br />
Apart from this dominant species, waste pile number<br />
3 also displays a mixture of various species in terms<br />
of preference for the environmental conditions. Thus,<br />
xerophile species such as Tamarix ramosissima live<br />
together with hygrophile species such as Salix fragilis<br />
and Salix matsudana. We can also encounter<br />
spontaneous Salix caprea (mesophile) on the<br />
phosphogypsum waste pile.<br />
The species of the genus Salix are dioicous, the<br />
sexes being separate: the trees bear m<strong>ale</strong> or fem<strong>ale</strong><br />
flowers. Some species of the genus Salix are<br />
interfertile. Different varieties of Salix alba and Salix<br />
fragilis crossbreed frequently in nature giving birth to<br />
different hybrids, among which the most common is<br />
Salix x rubens. The overlapping of the morphological<br />
features of the two species increases the <strong>de</strong>gree of<br />
difficulty in the correct <strong>de</strong>termination of the species<br />
[3]. Both the morphological studies and the genetic<br />
investigations realized on the S. alba – S. x rubens –<br />
S. fragilis complex indicate its division into two main<br />
groups. A group is ma<strong>de</strong> up of Salix alba and Salix x<br />
rubens, while the second group is ma<strong>de</strong> up of S.<br />
fragilis si S. x rubens var. basfordiana [4]. This<br />
division of the complex into the two groups concords<br />
with previous research (Triest et al., 1998, 2000),<br />
quoted by [4], who analyzed the isoenzymes and<br />
RAPD (Random Amplified Polymorphic DNA). As a<br />
result of these tests, the S. alba – S. x rubens – S.<br />
fragilis complex was divi<strong>de</strong>d into two groups: “S.<br />
alba-like” and “S. fragilis-like”.<br />
ISSN-1453-1267 © 2010 Ovidius University Press
The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010)<br />
The great variety of hybrids existing in nature<br />
prevents sometimes the exact i<strong>de</strong>ntification of the<br />
species of the genus Salix only by morphological<br />
features. It is possible that the specimens i<strong>de</strong>ntified<br />
on the phosphogypsum waste pile as belonging to the<br />
species S. fragilis, might be hybrids that inherited<br />
from the genitors the capacity to live on a salty<br />
substrate which lacks organic matter but has high<br />
humidity. Unfortunately, the lack of financial means<br />
prevented the accomplishment of ADNcp (ADN<br />
chloroplastic) analyses that allow the precise<br />
i<strong>de</strong>ntification of the species or supposed hybrids used<br />
within the experimental project for the setting up of a<br />
willow culture. The large number of hybrids of<br />
species of the genus Salix offers them increased<br />
capacities to adapt to the most diverse environmental<br />
conditions.<br />
Starting from this theory, the experimental<br />
culture using species of the genus Salix on the<br />
phosphogypsum waste pile seeks to i<strong>de</strong>ntify a species<br />
or a hybrid that, in the living conditions of the waste<br />
pile, should provi<strong>de</strong> the most consi<strong>de</strong>rable quantity of<br />
wood mass. I mention that the species Salix alba is<br />
newly introduced in the ecosystem of the<br />
phosphogypsum waste pile with the purpose of<br />
monitoring the production of biomass reported per<br />
surface unit.<br />
2. Material and Methods<br />
The experimental culture of Salix sp. on<br />
phosphogypsum waste pile no. 3 belonging to the<br />
S.C. Fertilchim – Marway S.A. company was set up<br />
on a vegetation-free surface of 540 square meters.<br />
For this purpose, Salix alba and Salix fragilis cuttings<br />
were used. They were collected from an area adjacent<br />
to the town of Navodari. Some Salix fragilis cuttings<br />
were collected from the trees that grew spontaneously<br />
on the waste piles. This surface was planted with 110<br />
cuttings belonging to the species S. alba (44 pieces)<br />
and S. fragilis (6 pieces). The cuttings were planted<br />
on parallel rows with a length of 20 m. The distance<br />
between two successive rows is three meters, while<br />
the distance between the cuttings on the same row is<br />
two meters. The number of cuttings on a row is<br />
eleven. The planting <strong>de</strong>pth is between 0.7 and one<br />
meter. Thus, two rows with S. fragilis were planted at<br />
186<br />
0.7 meters and eight rows (four with S. alba and four<br />
with S. fragilis) were planted at a <strong>de</strong>pth of one meter.<br />
The collection of cuttings occurred between 10-20<br />
March 2010, while<br />
their planting took place between 16-27 March 2010.<br />
Some of the cuttings (22 pieces) were collected from<br />
S. fragilis grown spontaneously on the<br />
phosphogypsum pile, while the others (88 pieces) –<br />
44 S. alba and 44 S. fragilis – were collected from<br />
the area adjacent to the town of Navodari. The age of<br />
the cuttings is between one and three years, while<br />
their sizes vary between one and 2.5 meters.<br />
In or<strong>de</strong>r to verify the influence of the<br />
microclimate effect, five rows of cuttings were<br />
planted in phosphogypsum ditches. The <strong>de</strong>pth of the<br />
ditch was approximately 0.4 meters, while the width<br />
was 0.3 meters. The planting <strong>de</strong>pth in four of the five<br />
ditches was measured to be one meter, taking the<br />
phosphogypsum surface as marker and not one meter,<br />
taking the bottom of the ditch as marker. In the case<br />
of the fifth ditch, the planting <strong>de</strong>pth is 0.7 meters and<br />
it was measured the same as in the previous rows.<br />
The planting method is presented in Figure 1.<br />
Fig.1. The way in which the cuttings were planted<br />
The other five rows that make up the witness<br />
area for the study of the microclimate influence were<br />
planted directly on the phosphogypsum surface,<br />
without digging ditches. The planting <strong>de</strong>pth in the<br />
case of four out of five rows is one meter, while a<br />
row was planted at 0.7 meters.
Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010)<br />
No preparation or maintenance works were done<br />
before and after planting the newly established<br />
culture (e.g. fertilization, irrigation, etc).<br />
3. Results and Discussions<br />
The monitoring of the growth and <strong>de</strong>velopment<br />
of the two willow species used to set up the<br />
experimental culture on the phosphogypsum waste<br />
pile, namely S. alba and S. fragilis, led to surprising<br />
results. Thus, on April 8, 2010, twelve days after the<br />
planting, it was observed that 107 out of the 110<br />
planted cuttings took root and sprouts had <strong>de</strong>veloped<br />
on them, while the first two-three leaves had already<br />
emerged in a small number of these cuttings. Three of<br />
the 110 cuttings did not take root and dried out. Two<br />
of these belong to S. fragilis and one to S. alba. On<br />
April 18, 2010, the sprouts on the 107 cuttings<br />
opened and the first leaves emerged. Some of them<br />
even grew three-four cm long shoots. On May 20,<br />
2010, it was observed that of the 107 cuttings that<br />
bore sprouts and shoots only 81 <strong>de</strong>veloped normally,<br />
with 5-20 cm long shoots. The other 26 were<br />
stagnating. The same situation was encountered on<br />
June 1, 2010, with the specification that the 81<br />
cuttings with normal <strong>de</strong>velopment had 10-35 cm long<br />
shoots and some of the 26 stagnating cuttings began<br />
to dry.<br />
We must mention that on the two rows (one with<br />
ditch for the verification of the microclimate<br />
influence and one without ditch, as witness), where<br />
the planting <strong>de</strong>pth was 0.7 meters, only S. fragilis was<br />
used. These two rows registered the highest number<br />
of stagnating cuttings about to get dry. The<br />
conclusion is thus that the planting <strong>de</strong>pth is very<br />
important, the greater the <strong>de</strong>pth, the more chances the<br />
cuttings have to take root and <strong>de</strong>velop normally. By<br />
taking phosphogypsum samples from various <strong>de</strong>pths<br />
and performing humidity analyses, it was observed<br />
that humidity increases directly proportionally with<br />
the <strong>de</strong>pth of the sample. Moreover, by analyzing<br />
samples from the surface of the phosphogypsum (0-5<br />
cm) and those from the bottom of the ditches (40 cm),<br />
it was noticed over a period of several months that<br />
the samples from greater <strong>de</strong>pths contained more<br />
water even though the months when the sample was<br />
collected were poor in precipitations. We specify that<br />
187<br />
no samples for the humidity test were collected in<br />
December 2009 because it rained on the day<br />
scheduled for the sample collection (December 12,<br />
2009). The graph in Fig. 2 presents the variation of<br />
humidity <strong>de</strong>pending on the time and <strong>de</strong>pth for the<br />
sample collection.<br />
Fig. 2. The variation of humidity <strong>de</strong>pending on the<br />
time and <strong>de</strong>pth of the sample collection<br />
This situation explains to a large extent the<br />
surprising presence of certain xerophyte species<br />
alongsi<strong>de</strong> hygrophyte ones on the phosphogypsum<br />
waste pile.<br />
In or<strong>de</strong>r to make a correct estimation of the<br />
rooting and normal <strong>de</strong>velopment of the cuttings<br />
<strong>de</strong>pending on species, we will only take into account<br />
the eight rows on which the cuttings were planted at a<br />
<strong>de</strong>pth of one meter (four with ditch for the<br />
verification of the microclimate effect and four<br />
without ditch, as witness). These eight rows inclu<strong>de</strong><br />
four rows planted with S. alba and four rows planted<br />
with S. fragilis. The total number of cuttings on these<br />
eight rows is 88, of which S. alba – 44, and S. fragilis<br />
– 44. Of the 44 S. alba cuttings planted on four of the<br />
eight rows, 38 <strong>de</strong>velop normally, while of the 44 S.<br />
fragilis cuttings planted on four of the eight<br />
rows, only 32 <strong>de</strong>velop normally. Though it is<br />
premature to draw pertinent conclusions, we can say<br />
that S. alba seems to be better adapted to the<br />
conditions of the phosphogypsum waste pile,<br />
consi<strong>de</strong>ring that its percentage of rooting and<br />
<strong>de</strong>velopment is 86.36%, compared to S. fragilis<br />
whose percentage is 72.72%. Taking these results
The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010)<br />
into account, it is surprising why S. alba did not<br />
emerge spontaneously on the phosphogypsum waste<br />
pile, consi<strong>de</strong>ring that we i<strong>de</strong>ntified a fem<strong>ale</strong> specimen<br />
from this species located at less than one km from the<br />
pile. This observation represents another argument in<br />
favor of the hypothesis that the species<br />
S. fragilis and S. matsudana existing on the<br />
phosphogypsum waste pile occurred by vegetative<br />
reproduction and not sexual one (from seeds).<br />
In regards to the lower rooting and <strong>de</strong>velopment<br />
<strong>de</strong>gree, it is probable that the age of the cuttings used<br />
for planting had an important role in this aspect.<br />
Thus, all the S. alba cuttings were young (un<strong>de</strong>r one<br />
year old), while those of S. fragilis were ol<strong>de</strong>r<br />
(between two and three years old).<br />
The phosphogypsum on waste pile no. 3,<br />
belonging to the S.C. Fertilchim – Marway S.A.<br />
company, contains 90% calcium sulfate or gypsum<br />
hydrated with water molecules (CaSO4x2H2O),<br />
alongsi<strong>de</strong> which we can encounter phosphorus<br />
pentoxi<strong>de</strong> (P2O5), traces of fluorhidric acid (HF),<br />
silicate (SiO2) and high concentrations of heavy<br />
metals [5]. An analysis bulleting released by the<br />
Constanta County Office for Agronomical Studies<br />
and Pedology on May 6, 2009 attests to the fact that<br />
the analyzed phosphogypsum contains no organic<br />
matter (humus), nor nitrogen (N). The nutrients<br />
contained by the phosphogypsum are potassium (K)<br />
in very low quantity and a higher amount of<br />
phosphorus (P), a fact also <strong>de</strong>monstrated by the<br />
analyses accomplished by the method of extraction<br />
with lactate acetate (A.L.), which were realized in the<br />
Pedology Laboratory of the Faculty for Natural and<br />
Agricultural Sciences within “Ovidius” University.<br />
Even though it is hard to believe that there are species<br />
that can <strong>de</strong>velop normally on a 100% mineral<br />
substrate, these four willow species (S. fragilis, S.<br />
matsudana and S caprea – spontaneous, and S. alba<br />
– introduced artificially) contradict this statement.<br />
Another factor that makes possible the normal<br />
<strong>de</strong>velopment of these species directly on<br />
phosphogypsum is their resistance in conditions of<br />
high soil salinity. Thus, S. fragilis, S. matsudana and<br />
S. seringeana tolerate high salinity values [6], while<br />
S. alba is “the most tolerant of all willow species to<br />
brackish water” [7]. In parallel, pH analyses were<br />
accomplished on samples collected from <strong>de</strong>pths<br />
between 5 and 100 cm which displayed pH values<br />
188<br />
between 4.7 and 6.56. These results, corroborated<br />
with the fact that the quoted species prefer a slightly<br />
acid pH, make the phosphogypsum waste pile a<br />
favorable environment for the setting up of a willow<br />
culture.<br />
As the graph in Fig. 3 shows, no correlation can<br />
be ma<strong>de</strong> between pH value and the <strong>de</strong>pth of sample<br />
collection.<br />
Fig. 3. The pH variation <strong>de</strong>pending on the time and<br />
<strong>de</strong>pth of the sample collection<br />
The only plausible explanation regarding this<br />
random distribution of the pH values in the<br />
phosphogypsum <strong>de</strong>posit could be that at the moment<br />
when the phosphogypsum suspension was<br />
neutralized, the milk of lime used did not always have<br />
the proper concentration.<br />
Willow is one of the well studied plants in or<strong>de</strong>r<br />
to use it in the phytoreparation processes, as it has a<br />
high capacity to accumulate heavy metals and it is<br />
easy to cultivate (Tremela et al. 1997; Pulford and<br />
Watson 2003) quoted by [8]. By concentrating<br />
important quantities of heavy metals in the shoots that<br />
will be collected every year, the willows will<br />
accomplish a <strong>de</strong>pollution of the phosphogypsum and<br />
this will be a first step towards its transformation into<br />
organic-mineral fertilizer when the willow plantation<br />
will be eliminated. The strong bioaccumulation<br />
phenomenon in the species of the genus Salix will be<br />
favored by the slightly acid pH and will accelerate the<br />
cleansing of the phosphogypsum [9].
Matei Lucian / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010)<br />
The harvesting should be done between<br />
November-February, after the leaves fall from the<br />
shoots. For harvesting, Claas Jaguar 880 GBE 1 or<br />
Claas Jaguar combines fitted with a HS2 harvesting<br />
head will be used. This type of combines transform<br />
the harvested shoots into a hash [10] that is left to dry<br />
and is then used in thermal power stations especially<br />
adapted for this solid fuel. The hash can be<br />
transformed into pellets used in regular thermal<br />
power stations as solid fuel.<br />
It is premature to speak about the role of the<br />
microclimate. By observing the phenotypical<br />
<strong>de</strong>velopment of the willows planted in ditches and by<br />
comparing them to the willows planted directly on<br />
phosphogypsum, no major differences were noticed<br />
in regard to the length of the shoots and the plant<br />
vigor. In the case of the number of cuttings that<br />
display normal <strong>de</strong>velopment, there are however small<br />
differences. Thus, in the case of S. fragilis, on the<br />
rows planted in ditches, there is a number of 17<br />
cuttings that <strong>de</strong>velop normally, compared to only 15<br />
cuttings with normal <strong>de</strong>velopment that were planted<br />
directly on phosphogypsum (witness area). In the<br />
case of S. alba on the rows planted in ditches, 20<br />
cuttings <strong>de</strong>velop normally, compared to only 17<br />
cuttings with normal <strong>de</strong>velopment planted directly on<br />
the phosphogypsum (witness area). It was noticed<br />
that the microclimate effect created by the ditches<br />
into phosphogypsum have a very important role in the<br />
case of seed germination and <strong>de</strong>velopment of the<br />
annual herbaceous species. Thus, a few months after<br />
the digging of the ditches, a large number of<br />
herbaceous plants <strong>de</strong>veloped on their bottom. These<br />
plants germinated from seeds brought by the wind,<br />
mostly belonging to the dominant species in the waste<br />
pile phytocoenosis, Puccinellia distans.<br />
4. Conclusions<br />
In or<strong>de</strong>r to set up a willow culture, it is best to<br />
harvest and plant the cuttings between February 15 –<br />
March 15.<br />
The planting <strong>de</strong>pth is very important. It was<br />
observed that at <strong>de</strong>pths exceeding 40 cm,<br />
phosphogypsum always displays humidity over 25%<br />
even if the sample was collected after a period with<br />
189<br />
no precipitations. The optimum planting <strong>de</strong>pth is 100<br />
cm.<br />
The age of the cuttings has a very important role<br />
in the rooting process and their normal <strong>de</strong>velopment.<br />
Thus, in the case of the one-year-old cuttings, the<br />
success percentage was higher.<br />
Over the entire surface of the waste pile, between<br />
0 and 100 cm, the distribution of the pH values is<br />
purely random and they range between a minimum of<br />
4.7 and a maximum of 6.56. This fact<br />
favors the <strong>de</strong>velopment of species of the genus Salix,<br />
which prefer a substrate with slightly acid pH.<br />
The total lack of organic matter and of nitrogen<br />
from the substrate does not prevent the species from<br />
the genus Salix to <strong>de</strong>velop normally, but it is possible<br />
to lead to a lower quantity of wood mass per surface<br />
unit.<br />
Even though within the experimental culture<br />
there was a larger number of cuttings of the species S.<br />
alba with normal <strong>de</strong>velopment, it is premature to say<br />
that this species is better adapted to the<br />
environmental conditions than S. fragilis, a<br />
spontaneous species in the phosphogypsum waste pile<br />
ecosystem.<br />
It is also premature to draw a conclusion about<br />
the influence of the microclimate generated by the<br />
ditches into phosphogypsum on the <strong>de</strong>velopment of<br />
the S. alba and S. fragilis cuttings. However, it was<br />
noticed that the microclimate generated by the ditches<br />
has a beneficial influence on the species of annual<br />
plants. Thus, in an interval of three months, a large<br />
number of herbaceous plants emerged on the bottom<br />
of the ditches, most of them belonging to Puccinellia<br />
distans, a dominant species in the phytocoenosis of<br />
the phosphogypsum <strong>de</strong>posit.<br />
The advantages of a culture with species<br />
belonging to the genus Salix on the phosphogypsum<br />
waste piles are multiple:<br />
- To obtain ecological fuel – the carbon<br />
eliminated by burning represents the carbon<br />
- Stored previously by photosynthesis, so no extra<br />
amounts of carbon are released into the<br />
atmosphere;<br />
- To use fields otherwise improper for other<br />
cultures and transform thus losses into profit;<br />
- The <strong>de</strong>velopment of the root system and of the<br />
willow shoots will prevent wind erosion;
The agricultural potential... / Ovidius University Annals, Biology-Ecology Series 14: 185-190 (2010)<br />
- Taking into account the fact that the harvest of<br />
shoots takes place between November and<br />
February, the annual leaf litter on the<br />
phosphogypsum surface will accelerate the<br />
process of soil formation;<br />
- By the phenomenon of bioaccumulation, the<br />
trees (shrubs) from the plantation will<br />
concentrate into their own structures important<br />
quantities of heavy metals that will be removed<br />
annually by cutting the shoots and <strong>de</strong>creasing<br />
thus the polluting content of the<br />
phosphogypsum;<br />
- The species of the genus Salix, fond of<br />
humidity, will retain part of the water resulted<br />
from precipitations, reducing drastically the<br />
levigation phenomenon and the draining of the<br />
phosphorus into the ground water;<br />
- The photosynthesis and evapo-transpiration<br />
generated by the willows will improve air<br />
quality and the local microclimate during the<br />
warm season.<br />
The main disadvantage is the fact that, being a<br />
monoculture, it will be more vulnerable to pests.<br />
Another possible disadvantage is that, because the<br />
cuttings are not planted like in a culture on swampy<br />
or irrigated land (the same <strong>de</strong>nsity per square meter),<br />
the quantity of wood mass per ha can be reduced.<br />
5. References<br />
[1]. SÂRBU I., Stefan N., Ivănescu Lăcrămioara,<br />
Mânzu C., 2001. Flora ilustrată a plantelor<br />
vasculare din estul României, Determinator, vol.<br />
I <strong>şi</strong> II, Editura Universităţii „Alexandru Ioan<br />
Cuza”, Ia<strong>şi</strong>.<br />
[2]. GOMOIU M.-T., Skolka M., 2001. Ecologie -<br />
Metodologii pentru studii ecologice, Ovidius<br />
University Press, Constanţa.<br />
[3]. SKVORTSOVA. K., 1999. Willows of Russia<br />
and adjacent countries. Taxonomical and<br />
geographical review. Univ. Joensuu Fac.<br />
Mathem. and Natru. Sci. Rept. Ser. 39. 307 pp.<br />
[4]. www.bfafh.<strong>de</strong>/inst2/sg-pdf/52_3-4_148.pdf.<br />
Diversity of dte willow complex Salix alba – S x.<br />
rubens – S. Fragilis<br />
190<br />
[5]. www.containment.fsu.edu/cd/content/pdf/466.pd<br />
f. Vegetative cover for phosphogypsum dumps:<br />
A Romanian field study.<br />
[6]. CROUCH R.J, Honeyman M.N., 1986, 'The<br />
relative salt tolerance of willow cuttings.' Journal<br />
of Soil Conservation, vol 42 (2), p. 103-104.<br />
[7]. ZALLAR S. Botanical Characteristics of the<br />
Willows, Soil Conservation Authority, Kew.<br />
[8]. www.sci.uszeged.hu/ABS/2006/Acta%20HP/50<br />
37.pdf. Change of root and rhizosphere characters<br />
of willow (Salix sp) induced by high heavy metal<br />
pollution.<br />
[9]. www.umass.edu/.../Phytoremediation%20PDF<br />
/PhytoLitReview.pdf. Phytoremediation literature<br />
review.<br />
[10]. www.bioeng.ca/pdfs/meetingpapers/2005/CSAE%20papers/05-080.pdf.<br />
Cutting, bundling and chipping shortrotation<br />
willow.