ACTA BIOLOGICA SLOVENICA VOL. 59 ŠT. 1 LJUBLJANA 2016 prej/formerly BIOLOŠKI VESTNIK ISSN 1408-3671 UDK 57(497.4) izdajatelj/publisher Društvo biologov Slovenije ABSACTA BIOLOGICA SLOVENICA VOL. 59 ŠT. 1 LJUBLJANA 2016 prej/formerly BIOLOŠKI VESTNIK ISSN 1408-3671 UDK 57(497.4) izdajatelj/publisher Društvo biologov Slovenije ACS ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 1-84 Acta Biologica Slovenica Glasilo Društva biologov Slovenije - Journal of Biological Society of Slovenia Izdaja - Published by Društvo biologov Slovenije - Biological Society of Slovenia Glavna in odgovorna urednica - Editor in Chief Alenka Gaberščik, e-mail: alenka.gaberscik@bf.uni-lj.si Tehnični urednik - Managing Editor Gregor Zupančič, e-mail: gregor.zupancic@bf.uni-lj.si Uredniški odbor - Editorial Board Robert Zorec (SLO), Matija Gogala (SLO), Alenka Malej (SLO), Livio Poldini (i), Mark Tester (AUS), Nejc Jogan (SLO), Mihael J. Toman (SLO), Franc Janžekovič (SLO), Branko Vreš (SLO), Boris Sket (SLO), Franc Batič (SLO), Hubert Potočnik (SLO), Georg A. Janauer (A), Doekele G. Stavenga (NL) Naslov uredništva - Address of Editorial Office Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Zasnova oblikovanja - Design Žare Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno - Printed on: 2016 Tisk - Print: Nonparel d.o.o., Medvode Naklada: 400 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega računa pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofinancirala Javna agencija za raziskovalno dejavnost Republike Slovenije Acta Biologica Slovenica je indeksirana v - is indexed in: CAB Abstracts, Web of Knowledge -Thomson Reuters ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 3-4 Am In memoriam: Mihael Bricelj (1946-2016) Mihael Bricelj oziroma Mišo za kolege in prijatelje ni bil zgolj mikrobiolog ampak zlasti vizionar, ki si je že dolgo pred časom zastavljal ključna raziskovalna vprašanja okoljske mikrobiologije. Šele današnje obdobje »omike« in »post-omike« bosta dali dokončne odgovore, ki jih je Mišo bežno razkril že pred desetletji. Svoje raziskovalno in pedagoško delo je začel kot asistent na Katedri za molekularno biologijo na Oddelku za biologijo Biotehniške fakultete. Študentje se ga spominjajo kot pedagoga, ki je še dolgo po uradnem zaključku vaj debatiral in razlagal novosti s področja genetike (takrat je bila genetika nov predmet, drugačen od klasičnih bioloških predmetov). Potrebe po mikrobiologu (bakteriologu) so ga pripeljale v skupino za lim-nologijo, ki je delovala na Nacionalnem inštitutu za biologijo. Tu je sodeloval pri raziskavah in monitoringih jezer in rek. Vzporedno je razvijal mikrobiološke metode sledenja kraških voda z bak-teriofagi in se pridružil mednarodnim projektom sledenja (Grčija, Avstrija) ter plodno sodeloval z Inštitutom za raziskovanje krasa Znanstvenoraziskovalnega centra SAZU. Bricljev raziskovalni opus kaže na njegovo široko raziskovalno zanimanje. Velik odtis je pustil v raziskovanju kraške hidrologije. Lahko bi rekli, da je bil pravzaprav prvi slovenski mikrobiolog, ki se je zavedal kompleksnega pomena in vloge mikrobov v kraških procesih. Njegova doktorska disertacija (1994) »Sledenje podzemnih voda z bakteriofagi bakterije Salmonella typhimurium« pa tudi ostalo raziskovalno delo s tega pomembnega področja kraške hidrologije, npr. sledenje s sporami in umetnimi sledili, sta dobila pomemben odmev ne samo v domači, ampak tudi v mednarodni raziskovalni srenji. Predaval je tudi na Tehniški Univerzi v Gradcu o bakteriofagih in hidrobiologiji v okviru mednarodne šole »Post Graduate Training Course on Groundwater Tracing Techniques«. Posebej ga je zanimalo stanje vodnih teles ter vpliv onesnaženja na mikrobioto in fitoplankton. Primarne produkcije ni mogoče razumeti brez dobrega poznavanja fototrofov, vključno mikroalg in cianobakterij. Prav zaradi tega je v soavtorstvu z Danijelom Vrhovškom, Gorazdom Kosijem, Brankom Vrešem in Tinetom Valentičičem objavil monografijo »Sladkovodne alge ali jih poznamo?« (1985). K mikroalgam se je z aplikativnega vidika ponovno vrnil v svojem zadnjem obdobju raziskovalnega ustvarjanja, ko je skušal v fotobioreaktorju z zeleno algo Chlorella vulgaris izboljšati biosintezo in akumulacijo lipidov, ki so osnova za t.i. »biodizel«. 4 Acta Biologica Slovenica, 59 (1), 2016 Za časa svojega raziskovalnega dela na Nacionalnem inštitutu za biologijo v Ljubljani (od 1972 do 2012) je bil priča različnim politikam, ki so ustvarjale in usmerjale raziskave, kar mu velikokrat ni bilo všeč in je to tudi jasno in temperamentno povedal. Opozarjal je na potrebo po usmerjenih raziskavah, vezanih na algologijo, sicer deficitarne biološke discipline v Sloveniji. Za razvoj algologije zgolj monitoring vodnih teles, ki vključuje le najosnovnejše in zakonsko določene parametre, brez dobre raziskovalne osnove, prav gotovo ni dovolj. Sicer pa svojega znanja ni ljubosumno hranil samo zase in ga širil zgolj preko ustaljenih »znanstvenih kanalov« v obliki znanstvenih člankov, ampak ga je posredoval tudi preko številnih strokovnih in poljudnih objav, saj mu številni tuji jeziki sploh niso bili »tuji«. Mogoče pa je bil še najpomembnej ši tisti neformalni način razširjenja njegovih znanj in informacij preko diskusij in t.i. »brain-storminga«. Rad je priskočil na pomoč zlasti mlajšim kolegom, ki so se šele začeli uveljavljati pri svojem raziskovalnem delu, saj je sam tudi iz svoje »pionirske« izkušnje vedel, da je pogosto pravi nasvet ob pravem času vreden več kot ducat prebranih znanstvenih člankov. Čeprav je Mišo dobro poznal biološke zakonitosti, ki ustvarjajo in vodijo življenje, in nekatere tudi sam odkril, je sam prehitro podlegel enemu najpogostejših vzrokov, ki jemlje človeška življenja. Mišo bi, kljub upokojitvi, še lahko marsikaj dodal v naše mozaično in pomanjkljivo znanje o življenju. To pa je sedaj popolnoma odvisno od njegovih naslednikov in vseh tistih, ki bodo vestno prebrali njegove prispevke o bioti in okolju, ter si tako priskrbeli inspiracijo za nadaljevanje zgodbe o življenju. Janez Mulec Gorazd Kosi Tina Eleršek ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 5-11 Am Germination rate of stinkwort (Dittrichia graveolens) and false yellowhead (D. viscosa) in relation to salinity Kaljivost smrdljive (Dittrichia graveolens) in lepljive ditrihovke (D. viscosa) v odvisnosti od slanosti Mateja Grašič, Sabina Anžlovar, Simona Strgulc Krajšek* University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana, Slovenia Correspondence: simona.strgulc@bf.uni-lj.si Abstract: This study was conducted to investigate the effect of salinity on germination rate of stinkwort (Dittrichia graveolens (L.) Greuter) and false yellowhead (D. viscosa (L.) Greuter). Lettuce (Lactuca sativa L.) was used as a positive control. Seeds of all three test species were sown on agar plates with three different NaCl treatments (2.5 g NaCl/L = 42 mM NaCl, 5 g NaCl/L = 85 mM NaCl and 10 g NaCl/L = 171 mM NaCl) and control treatment without NaCl. The three tested species germinated under all salinity conditions. However, they thrived best under control conditions and their germination rate was gradually declining with increasing salinity. Additionally, the start of their germination was delayed with increasing NaCl concentration. According to our findings, we can conclude that both Dittrichia species are very tolerant to salinity. Keywords: salinity, germination rate, tolerance, Dittrichia graveolens, stinkwort, Dittrichia viscosa, false yellowhead, Lactuca sativa, lettuce Izvleček: V raziskavi smo ugotavljali, kakšen je učinek slanosti na kaljivost smrdljive ditrihovke (Dittrichia graveolens (L.) Greuter) in lepljive ditrihovke (D. viscosa (L.) Greuter). Kot pozitivno kontrolo smo uporabili solato (Lactuca sativa L.). Semena vseh treh testnih vrst smo posejali na agarne plošče s tremi različnimi koncentracijami NaCl (2,5 g NaCl/L = 42 mM NaCl, 5 g NaCl/L = 85 mM NaCl and 10 g NaCl/L = 171 mM NaCl) ter kontrolnim tretmajem brez NaCl. Vse tri testne vrste so kalile pri vseh tretmajih, vendar je bilo njihovo uspevanje najboljše v kontrolnih razmerah, kaljivost pa je z naraščajočo slanostjo postopoma upadala. Poleg tega se je z naraščajočo slanostjo zamaknil tudi pričetek procesa kalitve. Na podlagi rezultatov lahko zaključimo, da sta obe vrsti ditrihovk zelo tolerantni na slanost. Ključne besede: slanost, stopnja kaljivosti, toleranca, Dittrichia graveolens, smrdljiva ditrihovka, Dittrichia viscosa, lepljiva ditrihovka, Lactuca sativa, solata Introduction Stinkwort (Dittrichiagraveolens (L.) Greuter) and false yellowhead (D. viscosa (L.) Greuter) are the only known species belonging to the genus Dittrichia, which is classified under the Aster-aceae family (Wraber 2010). They both originate in the Mediterranean region and can be found 6 Acta Biologica Slovenica, 59 (1), 2016 in Slovenia. However, only false yellowhead is considered to be indigenous in Slovenia, whereas stinkwort was only first discovered in 2008 and is regarded as an invasive alien species in this area (Frajman and Kaligaric 2009). The occurrence of false yellowhead in Slovenia is limited solely to its coastal part, where it is quite common in ruderal habitats (Wraber 2010, Jogan et al. 2001). Stinkwort has an even more intriguing distribution pattern, spreading only in ruderal habitats along highway (Frajman and Kaligaric 2009) together with some regional roads. The coastal region is greatly affected by the sea salt, whereas sites along main roads are frequently exposed to excessive application of ice-melting salts in the winter. Thus, we hypothesized that the occurrence of both Dittrichia species in Slovenia could be limited to sites with seemingly elevated salinity level. There exist various types of salts among which many are essential in terms of plant survival. Nonetheless, they quickly become detrimental in excessive amounts. One of the most fundamental and ubiquitous salts is NaCl. Soil salinity can be either primary, i.e. caused by natural processes, or secondary, that is human-induced by different human activities such as irrigation, fertilization and application of de-icing salts (Kotuby-Amacher et al. 2000, Parihar et al. 2015). Most plants cannot bear high salinity level as it causes water stress, ion toxicity, oxidative stress, nutritional disorders, alteration of metabolic processes, membrane disorganization, etc. (Hasegawa et al. 2000, Carillo et al. 2011). All these small-scale consequences reflect in their most vital processes, e.g. germination, growth and photosynthesis (Parihar et al. 2015) and can sometimes even lead to early senescence and death (Zhu 2007). Seed germination is believed to be the most critical phase of the plant life cycle. Therefore, species that are able to germinate under elevated salinity level have a substantial competitive advantage over species whose germination is entirely suppressed or at least delayed (DiTommaso 2004). Throughout evolution, some plant species have evolved different mechanisms to combat high salinity, for instance restriction of salt uptake, control of long distance transport of salt, extrusion of salt from the plants, compartmentalization of salt (Carillo et al. 2011, Parihar et al. 2015) and production of compatible solutes otherwise known as osmoprotectants (McNeil et al. 1999). So far, not many studies have discussed the relationship between either of the two Dittrichia species and salinity. Only a handful of studies have already reported about stinkwort being more sensitive to elevated salinity level (Ghorbanali et al. 2013) and the ability of false yellowhead to thrive in such conditions (Curadi et al. 2005). False yellowhead had already been proven to be tolerant to elevated salinity level (Flowers et al. 2012-2016) and to be able to survive in drastic salinity conditions with electrical conductivity values reaching up to 52 000 ¡¡S/cm (Curadi et al. 2005). Two other studies have focused more on the capability of stinkwort and false yellowhead to cope with dry conditions, where similarly false yellowhead did better than stinkwort (Oztürk and Mert 1983, Pérez-Fernández et al. 2006). Our major goal was to find out how increased salinity level affects germination of both Dittrichia species, as the existing distribution patterns of stinkwort and false yellowhead in Slovenia indicate to a potential competitive advantage of both species in habitats with elevated salinity level. Materials and methods We collected plant material from the following locations: - Flowering shoots of Dittrichia graveolens: Slovenia, Ljubljana, Roje, Obvozna cesta, gravel road bank, 46°6'17.17" N 14°28'54.36" E. Leg. & det.: S. Strgulc Krajšek & S. Anžlovar, 24. 9. 2013. - Flowering shoots of Dittrichia viscosa: Slovenia, Primorska, Koper, near the road exit from Istrska road to Ljubljanska street, along the fence enclosing a warehouse next to Planet Tuš commercial complex, ruderal site, 45°32'16.51'' N 13°44'7.38'' E. Leg. & det.: S. Strgulc Krajšek & S. Anžlovar, 10. 10. 2014. Lettuce seeds were bought from a local seed producer Agrina. Harvested plant material of both Dittrichia species was air-dried and stored separately in a dark and dry room until use. Only mature seeds were selected for this experiment. Before sterilising the seeds, we removed all seed appendices excluding pappus hairs. Seeds of Grašič et al.: Germination of Dittrichia in relation to salinity 7 all three test species were then surface sterilised by 15 min immersion in aqueous solution of sodium hypochlorite (16.5 g/l, Arekina, Šampionka Renče) and afterwards rinsed three times in distilled water for 5 min each time. To set the germination test, we first prepared 2% water-agar solutions, to which we added appropriate quantity of NaCl to get the desired NaCl concentrations (0, 2.5, 5 and 10 g NaCl/L). All test solutions were thereafter autoclaved and poured into 9-cm sterile Petri dishes. Seeds were placed on agar plates in a 5 x 5 cm array the following day. We used 3 or 4 replicates with 25 seeds for each tested combination. Agar plates were wrapped with transparent foil to limit evaporation and were stored at room temperature in good light conditions until the end of the experiment on its 25th day. Seeds were examined every day at roughly 24-hour intervals. A seed was considered germinated on the day of emergence of its radicle. We also monitored cotyledon opening as a marker of further seedling development. Data analysis was done using survival analysis in programme GraphPad Prism 5.01, which automatically compared curves representing different data sets (treatments) using Log-rank (Mantel-Cox) test. Along with germination tests, we also measured electrical conductivity of the soil from sampling locations of both Dittrichia species and electrical conductivity of the two test solutions with the highest NaCl concentrations. To avoid potential influence of any impurities while measuring electrical conductivity, ultrapure water (Milli-Q Plus 185 system) was used to prepare samples instead of distilled water. We repeated each measurement three times in 10-min intervals and ultimately expressed it as a mean value. Results Seeds of all three test species germinated under all salinity conditions (Figure 1: A, C, E). However, their germination rate was decreasing correspondingly with increasing NaCl concentration. Minor differences in this general trend could be due to numerous fungal infections in some Petri dishes. In addition to lowering final germination rate, increasing concentration of NaCl also delayed the sole process of germination in both Dittrichia species. As expected, lettuce reached the highest final germination rate of the three tested species (Figure 1: E). With the exception of the highest NaCl concentration, increasing salinity seemed to have a very negligible negative effect on its final germination rate. Nevertheless, the differences between 2,5 g NaCl/L and 5 g NaCl/L treatments and the control were statistically significant (P < 0.001). Final germination rate of stinkwort (Figure 1: A) and false yellowhead (Figure 1: C) in control treatment was very comparable. In general, ontogenesis was slow in both Dittrichia species, but considerably faster in false yellowhead compared to stinkwort. Although differences between treatments were a little less evident in false yellowhead than in stinkwort, they were still statistically significant in most cases. The alternation of dynamics of germination with increasing salinity was more pronounced in stinkwort than in false yellowhead. Cotyledon opening revealed similar findings as the process of germination (Figure 1: B, D, F), as the increasing NaCl concentration was slowing down the process of seedling development. In the case of D. graveolens in 10 g NaCl/L treatment, none of the tested seedlings developed to the phase with opened cotyledons (Figure 1: B). The percentage of D. viscosa seedlings with opened cotyledons in 5 and 10 g NaCl/L treatments was very low as well (Figure 1: D). Electrical conductivity of the soil from both sampling locations was relatively low and differed only slightly between both sampling locations (63.9 ^S/cm and 80.5 ^S/cm in Ljubljana and Koper, respectively). In contrast, high values of electrical conductivity were measured in our test NaCl solutions (9 000 and 18 000 ^S/cm in 5 and 10 g NaCl/L solutions, respectively), with the exception of control treatment (1 ^S/cm in 0 g NaCl/L solution). Discussion Overall, elevated salinity had a restraining effect on both germination of the seeds as well as cotyledon opening in seedlings, and was getting more and more intense with increasing NaCl 8 Acta Biologica Slovenica, 59 (1), 2016 A D. graveolens -•- control (dH2O) • ** 2,5 g NaCl/l *** 5 g NaCl/l *** 10 g NaCl/l H i t"" T T T ------ Time [days] D. viscosa " ns -•- control (dH2O) 2,5 g NaCl/l * -*-• 5 g NaCl/l * -♦-10 g NaCl/l Ty . Tj f T T---' j T-*............*................ ♦ ♦ »------ ------ ---------A . Time [days] D. graveolens -m- control (dH2O) ** 2,5 g NaCl/l * 5 g NaCl/l *** 10 g NaCl/l Time [days] D. viscosa -•- control (dH2O) ns 2,5 g NaCl/l • * -*-• 5 g NaCl/l 10 g NaCl/l . T '..... ......................-T . ! * ? ..... Time [days] B 100 100 80 80 60 60 = 40 40 O 20 20 0 0 0 10 20 0 10 20 C D 100 100 80 80 60 60 = 40 40 20 O 20 10 20 10 20 Figure 1: Germination rates and percentage of seedlings with opened cotyledons of Dittrichia graveolens (A, B), Dittrichia viscosa (C, D) and Lactuca sativa (E, F) in relation to salinity. The results of survival analysis done using Log-rank (Mantel-Cox) test, that compared each curve with corresponding control treatment, are shown in the legends of all graphs (***: P < 0.001, **: 0.001 < P < 0.01, * 0.01 < P < 0.05, ns: P > 0.05). Slika 1: Kaljivost semen in odstotek kalic z razprtimi kličnimi listi vrst Dittrichia graveolens (A, B), D. viscosa (C, D) in Lactuca sativa (E, F) v odvisnosti od slanosti. Rezultati preživetvenih testov, narejenih z uporabo Log-rank (Mantel-Cox) testa, s katerim smo primerjali vsako od krivulj s kontrolo, so prikazani v legendah ob vsakem grafu (***: P < 0,001, **: 0,001 < P < 0,01, * 0,01 < P < 0,05, ns: P > 0,05). Grašič et al.: Germination of Dittrichia in relation to salinity 9 concentration in all three test species, especially in stinkwort and false yellowhead. It was gradually slowing down their development and lowering the final percentage of germinated seeds and seedlings with opened cotyledons. Similar changes in patterns of germination dynamics under increasing salinity levels have also been observed by Lauchli and Grattan (2007). Along with having a direct negative impact on young plants, increasing NaCl concentration also hindered their development indirectly by affecting their roots. The roots in different NaCl treatments had dark root tips and less root hairs than the control seedlings. Many root tips have also turned upwards, as was already observed in a study by Levizou et al. (2002), where Lactuca sativa seeds were treated with Dittrichia viscosa extracts. Considering smaller differences in final germination rates between treatments and less variable developmental dynamics in false yellowhead compared to stinkwort, false yellowhead appeared to be more successful in dealing with elevated salinity level than stinkwort, which is in accordance with related preceding studies (Oztürk and Mert 1983, Curadi et al. 2005, Pérez-Fernández et al. 2006, Ghorbanali et al. 2013). In our case, final germination rate is not the best indicator of the sensibility of the tested plant species to increased salinity, as final germination rates of seeds treated with different NaCl concentrations did not differ much from the results of the control. The differences were much more visible in the following phases of seedling development, so the phase of cotyledon opening was more informative. It would be interesting to prolong the experiment to see further development, but for such purpose the experiment design should be different. According to USDA Agricultural Research Service, the threshold value that defines saline soils is 4 dS/m (Criteria for Diagnosing Saline and Sodic Soils 2006), which equals 4 000 ^S/ cm. Therefore, soil salinity of samples from both sampling locations was well below values that delineate non-saline soils from saline soils, whereas salinity of all of the tested NaCl solutions, even our lowest NaCl concentration (2.5 g NaCl/L), turned out to be above the aforementioned limit (except distilled water as control treatment). Regardless of the negative effect of elevated salinity level on the two Dittrichia species, our results have proven that both species, false yellowhead as well as stinkwort, are very tolerant regarding salinity. Their seeds were able to germinate and also mostly normally further develop in a remarkably wide range of NaCl concentrations, spanning from as low as 0 to the extreme 10 g NaCl/L (= 171 mM NaCl), while the majority of known plant species hardly grow or even cannot survive in such high salinity level as 100-200 mM NaCl (Carillo et al. 2011). Hence, according to our measurements of electrical conductivity, both Dittrichia species managed to handle even more than astonishing 200 times higher values of electrical conductivity compared to those that occur on their usual growing sites. Conclusions Apparently, elevated salinity level cannot be considered as the main factor in determining the occurrence of stinkwort and false yellowhead. It is presumably only one of many different factors that can also act in a hostile manner towards most plant species on growing sites with higher salinity level. For this reason, the distribution pattern of both Dittrichia species in Slovenia could be due to little competition on their growing sites and also their plasticity, which enables them to thrive on generally less favourable sites with many limitations. Povzetek Smrdljiva ditrihovka (Dittrichia graveolens) in lepljiva ditrihovka (D. viscosa) sta vrsti iz družine nebinovk (Asteraceae), ki imata v slovenskem prostoru različen status glede izvora. Lepljivo ditrihovko obravnavamo kot domorodno vrsto, ki pri nas uspeva le v primorski regiji (Jogan et al. 2001, Wraber 2010) v bližini morske obale oz., kjer je vpliv morja še zaznaven, medtem ko so pojavljanje smrdljive ditrihovke v Sloveniji prvič zabeležili šele leta 2008. Vse od takrat se hitro razrašča le vzdolž avtocestnega križa (Frajman in Kaligarič 2009) ter ob nekaterih večjih prometnicah. Glede na opisana vzorca razširjenosti obeh 10 Acta Biologica Slovenica, 59 (1), 2016 vrst smo domnevali, da obravnavani vrsti zelo dobro prenašata povišano slanost. K povišani slanosti na rastiščih lepljive ditrihovke najverjetneje prispeva morje, v primeru smrdljive ditrihovke pa se slanost tal poviša predvsem na račun zimskega soljenja avtocest. S to raziskavo smo skušali ugotoviti, kako povišana slanost vpliva na kalijovst in zgodnji razvoj kalic obeh vrst ditrihovk. Izvedli smo kalitvene teste na agarnih ploščah. Kot testno vrsto smo poleg obeh vrst ditrihovk uporabili še solato kot pozitivno kontrolo. Pripravili smo štiri različne koncentracije NaCl vključno s kontrolo, kamor smo dodali le destilirano vodo (0, 2,5, 5 in 10 g NaCl/L). Izmerili smo tudi elektroprevodnost vzorcev tal z nahajališč obeh vrst ditrihovk. Obe vrsti sta najbolje kalili v kontrolnem tret-maju (0 g NaCl/L). Višanje koncentracije NaCl je vse bolj zaviralo kalitev ter nadaljnji razvoj kalic. Določena semena do konca poskusa sploh niso vzklila, preostala pa so glede na kontrolo vzklila Literature bistveno kasneje. Zaviralen učinek soli je bil nekoliko bolj opazen na smrdljivi ditrihovki. Kljub vsemu se je izkazalo, da sta obe vrsti ditrihovk zmožni kaliti in uspevati pri povišanih koncentracijah soli, ki so tudi dvestokrat višje kot na njunih rastiščih v naravi. Prav zaradi tako širokega razpona uspevanja pri različnih koncentracijah soli ju v odnosu do slanosti lahko obravnavamo kot zelo tolerantni vrsti. Iz rezultatov lahko razberemo, da na vzorec razširjenosti smrdljive in lepljive ditrihovke v Sloveniji lahko vpliva tudi povišana slanost tal, saj prisotnost višje koncentracije soli na rastišču zmanjša konkurenčnost drugih vrst, ki nimajo tolikšne tolerantnosti na prisotnost soli kot obe vrsti ditrihovk. Acknowledgements This work was supported by Slovenian Research Agency grant no. P1-0212. Carillo, P., Annunziata, M.G., Pontecorvo, G., Fuggi, A., Woodrow, P., 2011. 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(http://www.sussex.ac.uk/affiliates/ halophytes/index.php?content=plantView&id=1867, datum dostopa: 18. 5. 2016) Frajman, B., Kaligarič, M., 2009. Dittrichia graveolens, a new alien species of the Slovenian flora. Hladnikia, 24, 35-43. Ghorbanali, M., Satei, A., Esmaeeli, M., 2013. Determining the optimum temperature and the effect of different salinity concentrations on germination and growth of the seedlings of Dittrichia graveolens (L.) Greuter. Plant and Ecosystem, 9 (34-1), 40-52. [abstr.] Hasegawa, P.M., Bressan, R.A., Zhu, J.-K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology, 51, 463-499. Jogan, N., Bačič, T., Frajman, B., Leskovar, I., Naglič, D., Podobnik, A., Rozman, B., Strgulc Krajšek, S., Trčak, B., 2001. Gradivo za Atlas flore Slovenije. Center za kartografijo favne in flore, Miklavž na Dravskem polju, 443 pp. Grašič et al.: Germination of Dittrichia in relation to salinity 11 Kotuby-Amacher, J., Koenig, R., Kitchen, B., 2000. Salinity and Plant Tolerance. Utah State University Cooperative Extension. (https://extension.usu.edu/files/publications/publication/AG-SO-03.pdf, datum dostopa: 18. 5. 2016) Lauchli, A., Grattan, S.R., 2007. Plant Growth and Development Under Salinity Stress. In: Jenks M. A., Hasegawa P. M., Mohan Jain S. (eds.): Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops. Springer, Dordrecht, pp. 1-32. Levizou, E., Karageorgou, P., Psaras, G.K., Manetas, Y., 2002. Inhibitory effects of water soluble leaf leachates from Dittrichia viscosa, on lettuce root growth, statocyte development and graviper-ception. Flora, 197, 2: 152-157. McNeil, S. D., Nuccio M. L., Hanson A. D. 1999. Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistance. 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In: Encyclopaedia of Life Sciences. John Wiley & Sons Ltd., Chichester (doi: 10.1002/9780470015902.a0001300.pub2, published online 15 January 2007). ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 13-21 Am The effects of leaf extracts of crack willow (Salix fragilis) on the growth of Japanese knotweed (Fallopia japónica) Vpliv listnih izvlečkov krhke vrbe (Salixfragilis) na rast japonskega dresnika (Fallopia japónica) Jasna Dolenc Koce University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana Correspondence: jasna.dolenc.koce@bf.uni-lj.si Abstract: Japanese knotweed (Fallopia japónica) is one of the most invasive of species in Europe, and can substantially reduce local native biodiversity. In the present study, the allelopathic potential of crack willow (Salix fragilis) on growth of Japanese knotweed was investigated. Aqueous extracts of 0.1% and 1% (w/v) were prepared from liophilised willow leaves and used for watering of young knotweed plants. Their growth was monitored for 196 days. Shoot height and leaf number were not affected but the mass of leaves and especially roots was reduced (up to 32%). At the end of experiment, biochemical characteristics related to physiological state (photochemical efficiency of PSII, protein content, enzyme activity of guaiacol peroxidase, lipid peroxidation) were measured. Mostly, they were at control levels, but the activity of guaiacol peroxidase and lipid peroxidation in roots increased. The extracts of crack willow showed moderate inhibitory effect on roots of treated knotweeds while the growth of shoots was unaffected. Given the root reduction described here, further studies with willow extracts and field studies with crack willow and Japanese knotweed plants would be reasonable. Keywords: Fallopia japónica, Salix fragilis, invasive species, leaf extract, allelopathy, growth Izvleček: Japonski dresnik (Fallopia japonica) sodi med najbolj inavzivne tujerodne vrste v Evropi in pomembno vpliva na zmanjševanje lokalne biodiverzitete. V raziskavi smo preučili alelopatski potencial krhke vrbe (Salix caprea) na rastjaponskega dresnika. Iz liofiliziranih listov vrbe smo pripravili 0,1 % in 1 % vodne izvlečke, s katerimi smo zalivali mlade rastline dresnika in spremljali njihovo rast 196 dni. Višina poganjkov in število listov sta bila podobna kot v kontrolnem tretmaju, medtem ko se je masa listov, predvsem pa korenin, močno zmanjšala (do 32 %). Ob koncu poskusa smo z biokemijskimi analizami (fotokemična učinkovitost FSII, vsebnost beljakovin, encimska aktivnost gvaiakol peroksidaze, lipidna peroksidacija) ocenili še fiziološko stanje rastlin. Večina izmerjenih lastnosti je bila na kontrolni ravni, razen peroksidazne aktivnosti in lipidne peroksidacije, ki sta se pri tretiranih rastlinah povečali. Izvlečki krhke vrbe kažejo zmeren alelopatski učinek na korenine tretiranih dresnikov, medtem ko so poganjki rasli neprizadeto. Glede na opisano zmanjšanje koreninskega sistema 14 Acta Biologica Slovenica, 59 (1), 2016 predlagamo nadaljnje raziskave z izvlečki vrbe in terenske raziskave o vplivu krhke vrbe na rast japonskega dresnika. Ključne besede: japonski dresnik, krhka vrba, invazivne vrste, listni izvleček, alelopatija, rast Introduction Japanese knotweed (Fallopia japonica (Houtt.) Ronse Decr., Polygonaceae) is among the 100 most invasive taxons in the world (Lowe et al. 2000) and is a well known and problematic invasive species in Slovenia, too. It grows in dense populations mainly in ruderal habitats and river banks. The main strategy for its spread in invaded regions is vegetative reproduction with rhizomes and stolons and high level of regeneration (Bailey et al. 2009). Sexual reproduction outside its natural range is limited but seed formation and germination were proved for Slovenian populations (Strgulc Kraj sek and Dolenc Koce 2015). So far, the mechanical removal and frequent cutting of knotweed plants have been the most common and efficient ways to limit their growth and spread. For the biological control of invasive plant species, allelopathy could be a potential mean. Allelopathy refers to chemical interactions among plants, including those mediated by microorganisms (Weston and Duke 2003) and is defined as supression of the growth and/or establishment of neighbouring plants by chemicals released from a plant or plant parts (Inderjit et al. 2011). These allelochemicals could be synthesized in all plant tissues and released in environment by leakeage, root exudates, evaporation and degradation of organic material (e.g. decomposed leaf litter). Majority of allelochemicals are secondary compounds; phenolic substances, flavonoids, terpens, alcaloids, steroids and function also in antimicrobial protection. Many plant species, including weeds and crops, have allelo-pathic activity, the most known and well studied are Juglans nigra, Ailanthus altissima, Alliaria petiolata, Centaurea maculosa (Weston and Duke 2003). Allelopathy could also be the mechanism which enables invasive plant species successful colonisation of new habitats; a mechanism known as a novel weapons hypothesis (Bais et al. 2003, Callaway and Aschehough 2000, Callaway et al. 2005, Hierro and Callaway 2003, Inderjit et al. 2011). Some studies on the allelopathic potential of knotweeds have already been carried out and biologically active compounds have been defined in this context (Dommanget et al. 2014, Fan et al. 2010, Gerber et al. 2008, Murrell et al. 2011, Vrchotova et al. 2007). As previously mentioned, river banks are often overgrown by Japanese knotweed and the same habitat type is characteristic for some species of willow (Salix). Willows have high physiological and ecological plasticity, they grow rapidly and can accumulate metals, and for these reasons they are commonly introduced in ecosystems for phytoremediation of degraded habitats (Alvarez et al. 2003, Kuzovkina and Quigley 2005, Prach and Pysek 2001). However, negative effects of willows on neighbouring plants (inhibited growth) were also reported due to shading, competition for mineral nutrients and allelopathy. At least 12 phenolic allelochemicals were detected in leaves of goat willow (Salix caprea) (Ikonen et al. 2002, Hallgren et al. 2003, Moohammadnor et al. 2010). In some areas, crack willow (Salix fragilis) is even considered invasive (Cremer 2003). It affects river ecosystems by releasing secondary compunds and organic matter (leaves, cork, fruits), shading, which all changes nutrient cycling, food chains and biodiversity (Groninger and Bohanek 2000, Doody and Benyon 2011). It has high level of regeneration and small fragments of shoots are dispersed by water to new locations where they quickly regenerate and start a new population (Budde et al. 2011). Leaf composition can also account for allelopathic potential of crack willow; leaves are compact and degrade slowly relasing tannins and phenolic compounds for a longer time (Julkunen-Tiitto 1985, Haapala et al. 2001). The aim of the present study was to evaluate the allelopathic potential of crack willow on invasive Japanese knotweed. Developmental Dolenc Koce: Allelopathic effect of crack willow on Japanese knotweed 15 changes during growth of young knotweed plants were observed and their physiological state was determined by measuring photochemical efficiency of PSII, protein content, activity of anti-oxidative enzyme guaiacol peroxidase and lipid peroxidation. Materials and methods Plant materials The mature leaves of crack willow (Salix fragilis L.) were collected in Ljubljana, Slovenia (46° 3' 55.63" N, 14° 27' 42.53" E) in October. Following their collection, the leaves were lyophi-lised and stored in dark at room temperature until the extracts were prepared. Young shoots of Japanese knotweed (Fallopia japonica var. japonica (Houtt.) Ronse Decr.) were collected in Ljubljana, Slovenia (46° 2' 59.84" N, 14° 28' 28.52" E) in April of the following year. The collected shoots had approx. 3 cm high aboveground stem and 2 cm long underground rhyzome with at least one stem bud. Each shoot was planted in a separate pot filled with mineral substrat vermiculite. The pots were kept in a climate chamber under control conditions of temperature (25 ±1 °C), humidity (40%), and light/ dark cycle (16h/8h; light intensity 160 ^M m"2s-1) for 2 months. After that, the plants were transplanted to pots with commercial garden soil for additional 4 months (196 days in total) and grown under laboratory conditions of ambient temperature, humidity and light intensity. Preparation of willow leaf extracts The lyophilised mature willow leaves were homogenised using a mortar and pestle. Following suspension of the ground leaves in distilled water (5 g leaves in 100 ml distilled water), the extraction was carried out for at least 24 h on a shaker (170 rpm; room temperature). The extracts were then vacuum filtered, once with filter paper for general use and twice with fine filter paper (388 Grade, 84 g/m2) to provide the 5% (w/v) aqueous extract that was frozen at -20 °C until needed. Defrosted extract was diluted in distilled water to give the final concentration of 0.1% and 1% (w/v). The extracts of final concentration were prepared fresh prior to watering the knotweed plants. Measuring of Japanese knotweed growth The knotweed plants were watered with distilled water as the control (N=5), and with the 0.1% and 1% aqueous willow extracts (N=10 for each treatment). In the first two months, the plants were watered with willow extracts once a month and in the following 4 months twice a month because their growth increased substantially. In the interim period, all knotweed plants were watered with distilled water every 3-4 days to prevent dehydration. Every week, the shoot height was measured and leaf number was counted to evaluate the growth dynamics. After 196 days of observation, the photochemical efficiency of the photosystem II was measured, and roots and shoots were separated, weighed, frozen in liquid N2, and stored at -20 °C prior to biochemical analyses. Photochemical efficiency The photochemical efficiency of PSII was measured on the 2nd or 3rd youngest leaf of the same size using modulated fluorometer PAM 2100 (Walz, Germany) according to Germ et al. (2005). After 15 min dark adaptation, leaves were illuminated with a saturating beam of white light (photosynthetic photon flux density = 8000 ^mol m-2 s-1, 0.8 s) to excite the fluorescence of chlorophyll a and the optimal quantum yield (Fv/ Fm) was detected. The effective quantum yield of PSII was measured by providing a saturating pulse of white light (PPFD = 9000 ^mol m-2 s-1, 0.8 s) using a standard 60° angle clip. Biochemical analyses Samples of ~100 mg roots and shoots/ leaves were homogenised in 1.5 ml of potassium phosphate buffer (100 mM, pH 7), centrifuged (20 817 g, 20 min, 4 °C) and the resulting supernatants were used to spectrophotometrically (UV-1800 Shi-madzu) determine the protein concentration (BCA Protein Assay Kits, Novagen), specific enzyme activity and MDA content (as the measure of lipid peroxidation), as previously described (Dolenc Koce et al. 2014) and as indicated briefly below. 16 Acta Biologica Slovenica, 59 (1), 2016 The activity of guaiacol peroxidase (G-POD; EC 1.11.1.7) was measured at 470 nm (e = 26.6 mM"1-cm"1). The reaction mixture contained 900 ¡l potassium phosphate buffer (50 mM, pH 7) with 1% guaiacol and 10 mM H2O2, with the addition of 100 ¡l of the supernatant samples described above. Lipid peroxidation was evaluated in terms of the content of malondialdehyde (MDA). Here, 200 ¡il of the supernatant samples obtained for the protein extraction described above was added to 800 ¡l acetic reagent (0.5% (w/v) thiobarbituric acid in 20% (w/v) trichloroacetic acid). The mixture was incubated for 30 min at 95 °C, and then chilled to stop the reaction. The MDA content was measured spectrophotometrically at 532 nm and 600 nm (Dolenc Koce et al. 2014). Statistical analysis Means and standard errors were calculated, and the samples were compared by t-test and ANOVA (Microsoft Excel, GraphPad Prism 3.02). The level of significance was set at p < 0.05. Results During the experiment which lasted cca. 6 months, some knotweed plants did not survive and final number of plants for statistical analysis was 4 for control, 8 for 0.1% extract and 10 for 1% extract treated plants. At the end of experiment, the roots of knotweed plants that were treated with willow leaf extracts were more affected than the aboveground stems and leaves. Shoot height and leaf number were at all measuring points similar as in control plants (Fig. 1). In case of 1% extract, the shoots were on average even heigher but the difference was not statistically significant. The root growth was evaluated at the end of the experiment by weighing the mass of the root system and was 21% and 32% lower in knotweed plants treated with 0.1% extract and 1% extract, respectively (Fig. 2). Despite the substantial decrease, the differences were not statistically significant (p = 0.148). Similar, but less inhibitory effect was observed with the total leaf mass which was approx. 20% lower in treated plants (p = 0.402). 0 20 40 6 0 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 Time (days) Time (days) Figure 1: Growth of knotweed plants treated with willow leaf extracts. A - shoot height, B - leaf number. Data are means (n = 4 for control, 8 for 0.1% extract and 10 for 1% extract treatment). Slika 1: Rast japonskega dresnika, tretiranega z listnimi izvlečki krhke vrbe. A - višina poganjka, B - število listov. Prikazane so povprečne vrednosti (n = 4 za kontrolo, 8 za tretma z 0,1 % izvlečkom in 10 za tretma z 1 % izvlečkom). Dolenc Koce: Allelopathic effect of crack willow on Japanese knotweed 17 Long-term treatment with willow extracts showed diverse and mainly statistically insignificant effects at the biochemical level (Tab. 1). Protein content increased up to 23% in roots and decreased up to 21% in leaves, the specific enzyme activity of G-POD incresed in all investigated tissues with maximal (313%) increase in roots, treated with 0.1% extract (ANOVA for G-POD in roots; p = 0.012). Lipid peroxidation, estimated as MDA content, was up to 76% higher in roots (ANOVA; p = 0.119) and up to 15% lower in leaves (ANOVA; p = 0.084). The increase of lipid peroxidation in roots correlates with decreased root biomass caused by treatment with willow extracts (Fig. 3). The negative correlation between these two parameters was higher (Pearson correlation coefficient r = -0.6353 and -0.1669 for 0.1% and 1% treatment) than in roots and leaves of control plants (for all root data r = -0.3562 and for all leaf data r = 0.0285). Figure 2: Mass of leaves, stem and roots of knotweed plants treated with willow leaf extracts. Data are means ± SE (n = 4 for control, 8 for 0.1% extract and 10 for 1% extract treatment). Slika 2: Masa listov, stebla in korenin japonskega dresnika, tretiranega z listnimi izvlečki krhke vrbe. Prikazane so povprečne vrednosti ± SN (n = 4 za kontrolo, 8 za tretma z 0,1 % izvlečkom in 10 za tretma z 1 % izvlečkom). Relatively good physiological state of leaves was confirmed also by results of photochemical efficiency of PS II, expressed as optimal quantum yield (F^Fro) which was not affected when willow extracts were applied (Tab. 1). Discussion Biological suppression of growth by allelo-pathic interactions is a possible mechanism to control and reduce spread of invasive plant species. Among most noxious species is Japanese knotweed which grows over river banks and anthropogeni-cally degraded habitats and reduces biodiversity (Bailey et al. 2009). In the present study, leaf extracts of crack willow were used as a source of potential allelochemicals because both species grow in the same habitat type and share growing conditions. When young knotweed plants were watered with willow extracts of two concentrations (0.1 and 1%), the mass of root system decreased and its physiological state was disturbed which was confirmed by increased biochemical parameters associated with oxidative stress. The activity of guaiacol-peroxidase which is the enzyme involved in the hydrogen peroxide degradation increased for more than 3-fold in roots and the content of malondialdehyde which is a product of peroxidation of membrane lipids increased up to 76%. Nevertheless, the aboveground shoots were less affected; the shoot height, leaf number, 18 Acta Biologica Slovenica, 59 (1), 2016 Figure 3: Correlation of malondialdehyde (MDA) content with mass of (A) roots and (B) leaves of knotweed plants treated with willow leaf extracts. Data are individual measurements per treatment (n = 4 for control, 8 for 0.1% extract and 10 for 1% extract treatment). Trendlines are linear regression lines. Slika 3: Korelacija med vsebnostjo malondialdehida (MDA) in maso (A) korenin in (B) listov pri japonskem dresniku, tretiranem z listnimi izvlečki krhke vrbe. Prikazane so posamezne meritve za tretma (n = 4 za kontrolo, 8 za tretma z 0,1 % izvlečkom in 10 za tretma z 1 % izvlečkom). Trendne črte so linearne regresijske premice. Table 1: Biochemical analysis and photochemical efficiency of knotweed plants treated with willow leaf extracts. Tabela 1: Biokemijska analiza in fotokemična učinkovitost japonskega dresnika, tretiranega z listnimi izvlečki krhke vrbe. Control 0.1% extract 1% extract Roots Protein concentration (^g/ml) 896.78 ± 168.87 982.94 ± 112.19 1102.68 ± 119.34 G-POD (^M/min*mg) 29.26 ± 3.22 120.96 ± 22.58* 63.35 ± 10.35 MDA (nM/g) 4.31 ± 0.92 7.60 ± 1.01 6.39 ± 0.73 Leaves Protein concentration (^g/ml) 2086.26 ± 343.73 1739.17 ± 259.02 1639.57 ± 205.50 G-POD (^M/min*mg) 85.68 ± 19.67 151.35 ± 24.02 127.93 ± 14.00 MDA (nM/g) 3.50 ± 0.24 3.34 ± 0.16 2.98 ± 0.11 Fv/Fm 0.82 ± 0.01 0.81 ± 0.01 0.81 ± 0.00 Data are means ± SE (n = 4 for control, 8 for 0.1% extract and 10 for 1% extract) G-POD, specific enzyme activity of guaiacol peroxidase; MDA, malondialdehyde content; Fv/Fm, optimal quantum yield Bold *, statistically significant difference between treated and control plants (t-test; p < 0.05). Dolenc Koce: Allelopathic effect of crack willow on Japanese knotweed 19 photochemical efficiency and lipid peroxidation were at control levels. On the other hand, the mass of leaves decreased and the activity of G-POD increased but the differences were not statistically significant. Different effects of Salix caprea leaf extracts were reported previously. The reduction of root mass was proved for seedlings of Picea abies treated with Salix caprea extracts (Schutt and Blaschke 1980). Also decreased germination of Arrhenaterum elatius, Lotus corniculatus and Plantago lanceolata was reported when seeds were directly treated with leaf litter of S. caprea but the effect was less inhibitory or even stimulatory when litter was added to substrate improving its quaility (Mudrak and Frouz 2012). The inhibition of growth can be related to some methodological problems as well. It was shown that type of substrate can influence the plant growth (Mudrak and Frouz 2012); when seedlings of tested plant species grew in sand, the allelo-pathic effects of willow were more pronounced because the sand has lower absorption capability and chemical interactions between compounds in plant extracts and sand are less intense than in soil. The same effect was observed in our study; when knotweed plants grew in mineral substrate vermiculite first 2 months, their growth was slow. When they were transferred to soil, their growth increased but the change was also related to different temperature, humidity and light conditions. To prove allelopathic potential of an extract, activated carbon can be added to the substrate to absorb chemicals (Prati and Bossdorf 2004). Also the use of distilled water for control could have negative effects on plants. The practical experiences show that watering with distilled water results in weaker growth than watering with tap water which contains higher level of minerals. The reduction of the underground tissues of Japanese knotweed could be important mechanism for limiting its growth and spread because rhyzome and stolons are the prime structures that enable knotweed successful vegetative reproduction (Bailey et al. 2009). To prove allelopathic potential of crack willow for biocontrol of Japanese knot-weed further studies are necessary using willow extracts of higher concentrations, root extracts, more frequent watering with extracts and field studies with co-growth of knotweed and willow plants in the same substrate. The latter is of special importance because in natural conditions other factors can influence allelopathic potential of certain plant species. Soil biota and chemistry, abiotic factors related to seasonal differences and neighbouring organisms (Inderjit et al. 2011) affect interactions among organisms therefore appropriate methodolgy for integrating chemically mediated interaction into ecology is crucial (Inderjit and Callaway 2003). In case of interactions between willows and knotweeds the allolopathic potential of knotweed to reduce willow growth should also be considered and tested. Conclusions Long-term experiments are important way to study allelopathic effects because they simulate conditions in natural environment. Our study shows moderate allelopathic effect of leaf extracts of crack willow to roots of Japanese knotweed while shoots developed unaffected. In roots, biomass decreased over 6 months of growth in the presence of willow extracts and biochemical characteristics related to oxidative stress elevated. Povzetek Japonski dresnik (Fallopia japonica) sodi med 100 najhujših invazivk v svetovnem merilu (Lowe et al. 2000). Njegova hitra rast in vegetativno razmnoževanje s korenikami in stoloni mu omogočajo uspešno naseljevanje novih habitatov, kjer zaradi svoje invazivnosti izpodriva avtohtone vrste in spreminja ekosisteme ter povzroča gospodarsko škodo. Njegovo odstranjevanje je večinoma kemično, kar lahko povzroča negativne posledice na ostale rastline in okolje na splošno, in mehansko. Eden od možnih biotičnih načinov zatiranj a bi lahko bila alelopatij a, tj. negativen vpliv ene rastline na drugo preko delovanja alelopatskih spojin, ki jih rastlina izloča ali sprošča v okolje in vključuje tudi delovanje preko mikroorganizmov, povezanih z rastlinami. V raziskavi smo želeli ugotoviti, ali vodni izvlečki iz listov krhke vrbe (Salixfragilis) vplivajo na rast in razvoj japonskega dresnika. Vrbe so vrste, ki se pogosto uporabljajo v poskusih stabilizacije in obnove uničenih ekosistemov. Poleg vlagoljubnih 20 Acta Biologica Slovenica, 59 (1), 2016 vrst najdemo med njimi tudi hitro rastoče pionirske rastline zgodnjih sukcesijskih faz, ki naseljujejo nove, s pomočjo človeka nastale odprte površine, torej podobna rastišča kot japonski dresnik. Poleg tega je zanje značilen tudi alelopatski potencial, ki skupaj s senčenjem in tekmovanjem za hranila zavira rast podrasti. Iz liofiliziranih listov krhke vrbe smo pripravili 0,1 in 1 % vodni izvleček, s katerim smo 1 do 2-krat mesečno zalivali mlade rastline japonskega dresnika, ki smo jih posadili v vermikulit in zemljo. Njihovo rast in razvoj smo spremljali 196 dni. Ugotovili smo, da so izvlečki zavrli predvsem razvoj korenin, kar se je pokazalo z zmanjšanjem njihove mase, povečale pa so se značilnosti, povezane z oksidativnim stresom, tj. lipidna peroksi-dacija, ocenjena kot vsebnost malondialdehida, in aktivnost antioksidativnega encima gvajakol peroksidaze. Poganjki so bili manj prizadeti, saj so bili višina poganjka, število listov, fotokemična učinkovitost FS II in lipidna peroskidacija na kontrolnih vrednostih. Zmanjšala se je masa listov, povečala pa aktivnost gvajakol peroksidaze, vendar so bile razlike statistično neznačilne. Slabša rast korenin zaradi delovanja izvlečkov iz listov krhke vrbe predstavlja temelj za nadaljnje raziskave, v katerih bi bilo potrebno preučiti delovanje koreninskih izvlečkov, izvlečkov z višjo koncentracijo, pogostejše zalivanje z izvlečki, ter ugotoviti kako vplivajo na rast japonskega dresnika vrbe, ki bi rastle skupaj z njimi v istem substratu. S tem bi lahko potrdili alelokemijsko delovanje krhke vrbe in njen potencial za biološko kontrolo invazivnih tujerodnih rastlin. Acknowledgments This study was supported financially by the Slovenian Research Agency, grant no. P1-0212. The author is thankful to Jona Šušteršič for technical assistance and dr. Tinka Bačič for valuable comments of the manuscript. 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Weed and crop allelopathy. Critical Reviews in Plant Sciences, 22, 367-389. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 23-32 Am Control of alpine dock (Rumex alpinus) by non-chemical methods Trajnostno odstranjevanje alpske kislice (Rumex alpinus) Urban Šilcab *, Marija Gregorib a ZRC SAZU, Institute of Biology, Novi trg 2, p.p. 306, SI-1000 Ljubljana, Slovenia bBiotechnical Centre Naklo, Strahinj 99, SI-4202 Naklo, Slovenia Correspondence: urban@zrc-sazu.si Abstract: Alpine dock (Rumex alpinus) is a troublesome weed particularly in protected zones or Natura 2000 areas, in which only non-chemical control measures can be applied. The aim of our study was to investigate by means of a field experiment the effectiveness of various non-chemical methods: mowing, manual removal, heating, foil and grazing by cattle and pigs. Floristic changes, cover, number of shoots and biomass were monitored at 14-day intervals for three consecutive years. Manual removal and foil were most successful, with almost complete removal of the biomass and cover of alpine dock, and mowing, which reduced the cover to 50%. Other methods were not as efficient. Animals avoid grazing on R. alpinus and heat merely supresses dock growth for a short period. Keywords: Slovenia, mountain pastures, agricultural management strategies, weed control, Rumex alpinus Izvleček: Alpska kislica (Rumex alpinus) je problematičen plevel gorskih pašnikov, še posebej v zaščitenih območjih ali območjih Natura 2000, kjer lahko uporabimo le nekemične metode zatiranja. S poljskim poskusom smo testirali učinkovitost različnih nekemičnih metod odstranjevanja alpske kisice: košnjo, ročno odstranjevanje, toploto, folijo in pašo goveda oz. prašičev. V 14- dnevnih obdobjih smo tri vegetacijske sezone spremljali floristične spremembe, pokrovnost, število poganjkov in biomaso. Najbolj uspešni sta bili metodi ročnega odstranjevanja in folija, kjer smo biomaso in pokrovnost kislice skoraj popolnoma odstranili, s košnjo pa smo ju zmanšali za polovico. Ostale metode niso bile tako uspešne. Živali se izogibajo alpski kislici, z ožiganjem pa smo rast kislice zaustavili le za krajši čas. Ključne besede: Slovenija, gorski pašniki, kmetijsko-upravljalske strategije, zatiranje plevelov, Rumex alpinus. Introduction Alpine dock (Rumex alpinus L.) is a perennial species consisting of a horizontal rhizome (and a root up to 300 cm long), above-ground vegetative shoots with three to five big leaves, and fertile stalks bearing smaller leaves and up to several thousand flowers and fruits (Kutschera and Lichtenegger 1992, St'astna et al. 2010). R. alpinus is a common plant species in all mountains of western, central 24 Acta Biologica Slovenica, 59 (1), 2016 and eastern Europe, including the Apennines, the mountains of the Balkan Peninsula and the Caucasus (Meusel et al. 1965). It builds species-poor monodominant stands and the cover ofR alpinus is often close to 100% (Kliment and Jarolimek 1995). The species is strongly nitrophilous and grows on nutrient-rich soils frequently near farm buildings. A high content of plant available potassium and nitrogen in soil favours R. alpinus (Bohner 2005). It can be regarded as a pasture weed, dominating and reducing valuable pasture areas. R. alpinus (as many grassland weeeds) has several harmful characteristics: low nutritive value, noxious (high oxalic acid content), avoided by animals, not suitable for conservation and high competitors occupying large areas (Bohner 2005, Dietl 1982, Vasas et al. 2015) Docks (Rumex spp.) are very troublesome weeds in agricultural land in many countries, in arable crops or permanent grasslands (Jeangros and Nosberger 1990), for short period only (Niggli et al. 1993). Non-chemical control of docks (Rumex spp.) has become important in recent years, mainly because of an increase in organic farming, and several non-chemical methods are applied: biological, mechanical and cultural. These consist of: frequent cutting, mechanical removal, heating, use of predators and parasites, grazing (Hejcman et al. 2014, Van Eekeren et al. 2006, Zaller 2004 and references cited there). The main aim is to hinder the build-up of seeds and weaken regrowth capacity by destroying biomass (Zaller 2004). In addition to organic farming, agriculture in Natura 2000 areas (in lowlands and mountains) requires management practices that do not rely on the use of herbicides and are beneficial for the conservation of species, habitats and the environment. In mountainous areas, agriculture is mainly Alpine dairy farming, with the animals grazed over the summer. Mountain pastures include a significant fraction of the plant species pool of Alpine regions (Batzing 1991), and well-managed pastures can support species-rich plant communities of high conservation value (Spatz 1975). Mountain pastures were often not properly managed in the middle of last century. One threat is abandonment or low management intensity, which leads to secondary succession and overgrowing of areas with shrubs and trees. The other is intensification by inappropriate grazing practices or use of fertilisers, which results in eutrophication and weed invasion (Galvanek and Janak 2008). The latter is responsible for the invasion of nitrophil-ous plant species (and R. alpinus as the most significant) and communities, which can spread over large areas and are not useful as pastures and farmers are not entitled to subsidies from agri-environmental-climate packages within the current Slovenia's Rural Development Programme (Dular et al. 2013). Another impact of alpine dock spreading in protected areas is a reduction of species diversity and the endangerment of rare plants (St'astna et al. 2010). The aim of our study was to investigate the use of various non-chemical methods on the control of R. alpinus in a mountain pasture under field conditions. To the best of our knowledge, only two field experiments for the control ofR. alpinus have been set up without the use of herbicides, but with fewer methods applied (Corradini and Artigianelli 1991) or monitoring was done only for one season (Tsarik 1987). Methods Study site The plot experiment was established in stands with 100% cover ofR. alpinus on the high mountain pasture Korosica (northern Slovenia) at an elevation between 1500 m - 1570 m (46.434348 N, 14.291490 E). Korosica is a part of the Karavanke Natura 2000 area (SI3000285). There are 56 ha of pasture land on Korosica and, according to the state prescribed pasture grazing order, 80 animals (cattle and horses) or 65 LU can graze from mid-June till mid-September. Alpine dock has spread abundantly, presumably due to excess stock or the use of mineral fertilizers during the last ca. 20 years, and now occupies 9% of the grazing area (Dular et al. 2013). The climate is cool and humid (Ogrin 1996). Average annual precipitation is 1680 mm (Podljubelj meteorological station), and average annual temperature is 3.6 °C (Krvavec meteorological station) (Anonymous 2014). Silc and Gregori: Control of alpine dock by non-chemical methods 25 Grassland vegetation on Korosica is classified as Homogyno alpinae-Nardetum Mraz 1956 - mat-grass acidophilous pastures of submontane to supramontane belts of mountain ranges and is a priority habitat type (Annex I habitat type 6230). Soil analysis was done before the set-up of the experimental plots in 2012. The analysis showed a strongly acidic soil (pH 4.2) with a low content of phosphorus (P: 20.18 mg/kg-1) and sufficient content of potassium (K: 283.79 mg/ kg-1). Soil samples were analysed by Agrochemical laboratory of the Agricultural Institute of Slovenia. Experimental design Testing of sustainable removal of R. alpinus on Korosica lasted three vegetation seasons (2012-2014). The removal experiment was set up as a random block on 4 x 4 m test plots (in 4 replicates) and two 10 x 15 m plots (in single versions). Various methods were tested: mowing, flaming, foil cover, manual removal and grazing (cattle and pigs). For Mowing, Heat, Foil and Manual, we used four replicates, while Cattle and Pigs were only tested in the first year, and using a larger fence, because of the difficulty of using the animals (we were unable to set larger plots and the number of animals was limited) and the remote location of the Alpine dairy farm for transport. Heat treatment was done with an open flame and plants were flamed until foliage was burned down. Plots were mowed every 14 days (during the vegetation season), starting mid-June (2012), and the biomass was removed. Docks were manually excavated in the first year and the roots were collected and removed from the plots. Black polyethylene foil was installed permanently for two seasons. Animals were enclosed in the two larger plots: cattle grazing - 6 cattle / 2h a day / through the whole season and pigs rooting - 2 pigs for 4 weeks. In the case of pigs, we used the only autochthonous breed in Slovenia (Krskopolje pig), which is adapted to be kept outdoor. After treatment of the plots, we sowed a commercial grass seed mixture (Trifolium repens 5%, Phleum pratense 16%, Lolium perenne 79%, a product of Semenarna (Ljubljana). Commercial seed mixture was used because site-adapted mixtures were not available. Data collection Vegetation was sampled according to the Braun-Blanquet (1964) method on 9 m2 plots. Relevés were made prior to the start of management and at the end of every vegetation season, after the different removal treatments. All data were sampled prior to the start of the experiment, except for biomass, and repeatedly at the end of every vegetation season. Above ground biomass was sampled on two subplots (0.5 x 0.5 m) within every sampling plot and only R. alpinus plants were cut. The biomass was air dried and then kept at 104 °C for 24h and weighed. Every 14 days, the height, cover estimation and number of shoots were sampled on two 1 m2 subplots. Cover was visually estimated in percentages. An individual shoot was defined as a group of leaves that clearly formed a separate shoot, although not necessarily from a different root. Data analysis All data were tested for normality and homogeneity of variance prior to testing the differences. ANOVA and Non-parametric tests were used in STATISTICA (StatSoft 2007). The floristic composition was compared in a matrix (plots by species) arranged in JUICE (Tichy 2002). De-trended correspondence analysis (DCA) was done in Canoco (ter Braak and Smilauer 2002). Species cover values in percentages were square root transformed. Results The association Rumicetum alpini Beger 1922 was fully developed before the experiment, with characteristic species R. alpinus and Stellaria nemorum dominating and with 100% cover of the site. Stands were species poor, with a few nitrophilous species (Urtica dioica) and species indicating of periodically wet soil being present (Deschampsia cespitosa, Ranunculus repens). Changes in floristic composition differed among treatments. The greatest changes in species composition were in plots covered by foil and those subjected to manual excavation (Fig. 26 Acta Biologica Slovenica, 59 (1), 2016 1). Dock plants were completely removed there and replaced with grassland species (Lolium perenne) from seed mixture. Changes were gradual on mowed plots, where species turnover was slower and alpine dock covered half of the plot after three years, although grasses were already developed. Other plots had a similar position on the DCA ordination graph, indicating smaller changes after treatments (Fig. 1). The species number per plot after three years (results not shown) increased in all treatments except for Foil but the highest increase was on mown plots. Comparisons within treatments showed significant differences in biomass between the first year and the following two for Manual, Foil and Mowing, while Heat, Cattle and Pigs did not differ from Control (Fig. 2). Differences in biomass calculated for the last sampling year 2014 (one-way ANOVA, Bonferroni test) showed that Heat, Cattle and Pigs did not significantly differ from Control plots. The treatments Manual, Foil and Mowing were significantly different from Control but were not significantly different from each other. Changes in the cover of R. alpinus within each treatment show a significant reduction of cover for Manual and Foil, while Mowing significantly reduced cover only after the second year (Fig. 3). Pigs were very successful in cover reduction but R. alpinus recovered in the third year because the treatment lasted for only one year. There was a similar result with the Cattle treatment. The height of the dock plants was already significantly lowered by the Manual and Foil treatments after the first year, while Mowing reduced it only in the last sampling year (Fig. 4). Other removal treatments were not successful in that. The height of plants was also very variable among sampling years, since this plant trait is related to seasonal climatic variables that differed during the experiment (e.g., very dry 2013 and wet 2014 season). The number of shoots was significantly lowered by Manual, Foil and Mowing (Fig. 4). The number also varied among years, since sampling of particular shoots is very subjective, although it was done by the same observer. Figure 1: DCA analysis of vegetation plots. Eigenvalues for the first four DCA axes are 0.390, 0.171, 0.128 and 0.100, respectively. Foil-full circle, manual-full quadrat, mowing-rectangle, heat-triangle, control-diamond, pigs-empty circle, cattle-empty quadrat. Slika 1: DCA analiza vegetacijskih ploskev. Lastne vrednosti prvih štirih osi so 0,390, 0,171, 0,128 in 0,100. Folija: poln krog, ročno odstranjevanje: poln kvadrat, košnja: pravokotnik, toplota: trikotnik, kontrola: diamant, prašiči: prazen krog, govedo: prazen kvadrat. Silc and Gregori: Control of alpine dock by non-chemical methods 27 a it ac i -r fr a i- Il a T 1 T„ b n lffl-fl J 1 Manual Foil Mowing Heat Control Cattle Pigs Management Figure 2: Box plot of biomass of R. alpinus on plots with different management in three consecutive years. Means with the same letter are not significantly different from each other (one-way ANOVA, P<0.05). Slika 2: Škatla z brki biomase alpske kislice na ploskvah z različnim načinom zatiranja v treh zaporednih letih. Povprečja, označena z isto črko, med seboj niso statistično značilno različna (eno-fakorska ANOVA, P<0,05). !• ! t Ta i Fi a j Fa r5 u a j M i a Jac ■ I b t A ill ii* Jbc Manual Foil Mowing Heat Control Cattle Pigs Management Figure 3: Box plot of cover of R. alpinus on plots with different management in three consecutive years. Means with the same letter are not significantly different from each other (one-way ANOVA, P<0.05). Slika 3: Škatla z brki pokrovnosti alpske kislice na ploskvah z različnim načinom zatiranja v treh zaporednih letih. Povprečja, označena z isto črko, med seboj niso statistično značilno različna (eno-fakorska ANOVA, P<0,05). 28 Acta Biologica Slovenica, 59 (1), 2016 ' bc ac 1» T" b I bd a n I - 1 i 1 3 Ta a' 1 f a a J a S 1 i 1 bd 1 Ï j. Ú ¿b T T j : áb É.fí j Manual Foil Mowing Heat Control Cattle Pigs Management Figure 4: Box plot of height of R. alpinus plants in plots with different management in three consecutive years. Means with the same letter are not significantly different from each other (one-way ANOVA, P<0.05). Slika 4: Škatla z brki višine alpske kislice na ploskvah z različnim načinom zatiranja v treh zaporednih letih. Povprečja, označena z isto črko, med seboj niso statistično značilno različna (eno-fakorska ANOVA, P<0,05). Manual Foil Mowing Heat Control Cattle Pigs Management Figure 5: Box plot of number of shoots of R. alpinus in plots with different management in three consecutive years. Means with the same letter are not significantly different from each other (one-way ANOVA, P<0.05). Slika 5: Škatla z brki števila poganjkov alpske kislice na ploskvah z različnim načinom zatiranja v treh zaporednih letih. Povprečja, označena z isto črko, med seboj niso statistično značilno različna (eno-fakorska ANOVA, P<0,05). Silc and Gregori: Control of alpine dock by non-chemical methods 29 Discussion Most research interest in floristic changes related to various management techniques has been oriented into changes of semi-natural grasslands in lowlands after changes in management or its cessation, and the impact of other Rumex species (R. obtusifolius and R. crispus). Studies dealing with Rumex spp. are in general dedicated to the removal of docks and less with the species composition of the plant community they invade. The only study monitoring floristic changes was made by Corradini and Artigianelli (1991). Re-sowing of grasses was important to establish a new plant community and to prevent R. alpinus from growing again and to suppress its competitive ability. It is known that the seed bank has a limited role in the restoration of degraded sites (Handlova and Munzbergova 2006). The use of autochthonous seed mixtures of potential vegetation types or use of hay from an identical plant community would facilitate the succession but they were not available. Although docks are troublesome weeds in grasslands, most control studies have been done on "lowland" species, e.g., R. obtusifolius and R. crispus (Van Eekeren et al. 2006, Zaller 2004). In addition, studies about alpine dock have often been partial, using particular methods (also chemical), or short term (see St'astna et al. 2010). The largest reduction of R. alpinus was achieved by mowing, foil covering and manual excavation. Regular and frequent mowing has already been reported to influence R. alpinus (Corradini andArtigianelli 1991, Hujerova et al. 2013, St'astna et al. 2010, Tsarik 1987). The frequency of cutting is the most important and, not surprisingly, the more frequent the cutting the more effective is the dock suppression. In our study, we used cutting every 14 days since this period has already been shown to be effective (Tsarik 1987, Zaller 2004). The reduction of cover and biomass by mowing was very gradual compared to foil and excavation. This is congruent with the findings of Courtney (1985) that even five to seven cuts reduced the abundance of dock by only 60 %. When stands are mown less frequently, seedling emergence and seedling survival until next year increases (Tsarik 1987). Nevertheless, successful suppression is possible through regular mowing and removal of the biomass (Zaller 2004), although not in a short time (Pignatti and Pignatti 2014). Combination of mowing and grass seeding proved to be successful, which is congruent with the findings of Corradini andArtigianelli (1991). The competition of grasses and herbs is not enough to restrict docks in the long term (Zaller 2004) and should be combined with some other management. It is important to cut dock at a height of 10 cm to enable other plants to regenerate faster than Rumex. Excavation successfully removed dock plants but it is a very time consuming method. Tillage is usually applied as the ultimate non-chemical control measure on heavy Rumex infestations on arable land, and also on grassland, although contrasting results are reported (see review by (Zaller 2004). We removed the upper soil layer with roots and since the rhizomes usually grow at a depth of up to 5 cm (Klimes 1992) or, less frequently, between 10-12 cm (Kliment and Jarolimek 1995), the removal of alpine docks was successful. It is necessary to remove and destroy the roots so they cannot regenerate. New plants can germinate from the remains of root fragments and from the seed bank (Tsarik 1987), so sowing grasses is important in order to suppress young plants that emerge. After three years, only a few small plants were present in the plots, identical to the results of Bucharová (2003), although she used herbicide. The use of foil (or any other covering material) to reduce the light to weeds or any other invasive species is common practice (Bond and Grundy 2001) and it has been successfully applied to R. alpinus (Bechtold and Machatschek 2011). Light availability is a crucial resource in R. alpinus stands and control through competition for light should be successful (Zaller 2004). After one year, the docks are destroyed because of light reduction and high temperatures under the foil in the summer period. Foil is less suitable for large areas, especially in mountains with unfavourable climatic conditions. Treatment with flame reduced the biomass but not the cover and the docks regenerated after the first year. Flame has been successfully used in dock infested grasslands but only on locally infested spots and on single plants (Potsch 2003), while this method was less suitable in large patches of R. alpinus, similar to results in R. obtusifolius 30 Acta Biologica Slovenica, 59 (1), 2016 (Zaller 2004). In the case of large patches, all plants were damaged by the heat and the herbs were unable to compete with the docks. Rumex species are rarely grazed by animals and alpine dock is avoided by cattle and horses but readily eaten by goats (Bohner 2005, Ellenberg 1996, Hejcman et al. 2014) and has been used as pig fodder in the past (Wendelberger 1971). In our study, we used cattle and pigs for only one season and this resulted in some suppression of the alpine dock cover but probably more as a result of cattle trampling and tillage by pigs than grazing. Trampling can also reduce the R. alpinus above ground biomass (Tsarik 1987). Goats and sheep or combined with cattle graze on docks and effectively remove plants from grasslands (Hejcman et al. 2014) but in the case of mountain pastures in SE Central Europe, grazing of sheep and goats is not common or is traditionally limited to certain localities (part of NW Julian Alps) and the introduction of new animals would require a change in grazing policy related to NATURA 2000. We must also stress that all grazing experiments from the literature were made in grasslands in which docks are scatter distributed, while alpine dock forms large monodominant stands and such patches are even more avoided by grazing animals. Acknowledgements We would like to thank Mitja Lazar, Gaj Ulčnik, Nejc Kavčič, Iztok Sajko and Petra Košir for help during field work. Martin Cregeen kindly checked our English. Project ALPA (Nature sustainable management of mountain pastures on protected areas (2012-2014)) was financed from the program EU, Objective 3 - European territorial cooperation, Operation program Slovenia-Austria 2007-2013. UŠ was financed through ARRS project P1-0236. References Bätzing, W., 1991. Die Alpen - Entstehung und Gefärdung einer Europäischen Kulturlandschaft. C.H. Beck, München, 287. Bechtold, J., Machatschek, M., 2011. Alpenampferbekämpfung durch Lictenzug. Der Alm- und Bergbauer, 4, 21-23. Bohner, A., 2005. Rumicetum alpini Beger 1922- species composition, soil-chemical properties, and mineral elemnt content. Wulfenia, 12, 113-126. Bond, W., Grundy, A. C., 2001. Non-chemical weed management in organic farming systems. Weed Research, 41(5), 383-405. Braun-Blanquet, J., 1964. Pflanzensoziologie. Grundzüge der Vegetationskunde. Springer Verlag, Wien, 865. Bucharova, A., 2003. Rumex alpinus L. v Krkonošich-rozšiufeni a management. Charles University of Prague, Dipl. thesis. 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Vegetation Mitteleuropas mit den Alpen. Ulmer Verlag, Stuttgart, 1095. Galvanek, D., Janak, M., 2008. Management of Natura 2000 Habitats. 6230 *Species-rich Nardus Grasslands. European Commission, http://ec.europa.eu/environment/nature/natura2000/manage-ment/habitats/pdf/6230_Nardus_grasslands.pdf. Handlova, V., Munzbergova, Z., 2006. Seed banks of managed and degraded grasslands in the Krkonose Mts., Czech Republic. Folia Geobotanica, 41(3), 275-288. Hejcman, M., Strnad, L., Hejcmanova, P., Pavlu, V., 2014. Biological control of Rumex obtusifolius and Rumex crispus by goat grazing. Weed Biology and Management, 14(2), 115-120. Hujerova, R., Pavlu, V., Hejcman, M., Pavlu, L., Gaisler, J., 2013. Effect of cutting frequency on above- and belowground biomass production of Rumex alpinus, R.crispus,R.obtusifolius and the Rumex hybrid (R.patienta x R.tianschanicus) in the seeding year. Weed Research, 53(5), 378-386. Jeangros, B., Nosberger, J., 1990. Effects of an Established Sward of Lolium perenne L. on the Growth and Development of Rumex obtusifolius L. Seedlings. Grass and Forage Science, 45(1), 1-7. Kliment, J., Jarolimek, I., 1995. The Rumex alpinus communities in Slovakia. Biologia, 50(4), 349-365. Klimes, L., 1992. The clone architecture of Rumex alpinus (Polygonaceae). Oikos, 63(3), 402-409. Kutschera, L., Lichtenegger, E., 1992. Wurzelatlas mitteleuropäischer Grünlandpflanzen, Bd. 2: Pteridophyta und Dicotyledoneae (Magnoliopsida) T. 1: Morphologie, Ökologie, Verbreitung, Soziologie, Wirtschaft. Gustav Fischer Verlag, Stuttgart, Jena, New York. Meusel, H., Jäger, E., Weinart, E., 1965. Vergleichende Chorologie der zentraleuropaeischen Flora. Gustav Fischer Verlag, Jena. Niggli, U., Nösberger, J., Lehman, J., 1993. Effects of nitrogen fertilization and cutting frequency on the competitive ability and the regrowth capacity of Rumex obtusifolius L. in several grass swards Weed Research, 33(2), 131-137. Ogrin, D., 1996. 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ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 33-44 Am Gold nanoparticles do not induce adverse effects on terrestrial isopods Porcellio scaber after 14-day exposure Nanodelci zlata nimajo negativnih učinkov na kopenske rake vrste Porcellio scaber po 14-dnevni izpostavitvi Pavlina Skarkovaa, Tea Romihb1*, Monika Kosb, Sara Novakb, Veno Kononenkob, Anita Jemecb, Milada Vavrovaa Damjana Drobneb Pavlina Skarkova and Tea Romih equally contributed to this work. aBrno University of Technology, Faculty of Chemistry, Purkynova 118, 612 00 Brno, Czech Republic bUniversity of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia Correspondence: tea.romih@seyens.com Abstract: Despite the anticipated environmental release of anthropogenic gold nanoparticles (AuNPs), there is currently not enough data on their potential impact on terrestrial environment. In the current study, we investigated the effects of considerably low concentrations of AuNPs on terrestrial isopods (Porcellio scaber) after 14 days of exposure. The effects on mortality, weight change, feeding rate, avoidance/preference feeding behavior, and cell membrane destabilization of digestive gland cells were followed. In parallel, the accumulation of Au in the digestive glands was measured. Our results show that none of the tested parameters was affected in isopods under given exposure doses (10 and 60 ^g Au/g dry leaf) and exposure duration. No Au was assimilated in the digestive glands. Also, the same doses of the reference chemical, AuCl3, showed no effect. We conclude that these concentrations of AuNPs are safe for terrestrial isopods. We encourage reporting the results showing no adverse effects of nanoparticles to balance the prevailing publication of their adverse effects. This will help to build a realistic public perception of the environmental risk of nanomaterials. Keywords: nanoparticles, Au3+, avoidance behavior, bioaccumulation, safety Izvleček: Kljub naraščujoči uporabi nanodelcev zlata (ND Au) trenutno še vedno ni dovolj podatkov o njihovih potencialnih negativnih učinkih na kopenske organizme. V tej študiji smo proučevali vpliv relativno nizkih koncentracij ND Au na kopenske rake vrste Porcellio scaber po 14-dnevni izpostavitvi. Proučevali smo vpliv na smrtnost, maso živali, stopnjo prehranjevanja, izogibalno vedenje in destabilizacijo membrane celic prebavnih žlez. Izmerili smo tudi asimilacijo Au v prebavnih žlezah. Rezultati so pokazali, da testirane koncentracije ND Au (10 in 60 |ig Au/g lista) pri kopenskih rakih 1 Current address: SEYENS Information Solutions and Education Ltd., Krimska ulica 20, 1000 Ljubljana, Slovenia 34 Acta Biologica Slovenica, 59 (1), 2016 niso vplivale na nobenega izmed testiranih parametrov po danem času izpostavitve. V prebavnih žlezah nismo izmerili Au. Enake rezultate smo dobili tudi z referenčno kemikalijo, soljo AuCl3. Zaključujemo, da so testirane koncentracije ND Au varne za kopenske rake. Naše mnenje je, da se na področju nanotoksikologije trenutno objavlja predvsem rezultate, ki kakorkoli nakazujejo na negativni vpliv nanodelcev na organizme. Zato želimo stimulirati tudi objavo rezultatov, ki kažejo nasprotno. Na ta način bomo pripomogli k bolj uravnoteženemu in realističnemu mnenju javnosti o okoljski varnosti nanomaterialov. Ključne besede: nanodelci, zlato, izogibalno vedenje, bioakumulacija, varnost Introduction Gold is a trace element in lithosphere, hydrosphere, and biosphere. It occurs naturally at concentrations around 4 |g/kg in the Earth's crust, <200 |g/kg in rocks, <2 mg/kg in soils, <1 |g/L in freshwaters and <5 |g/L in marine waters, while waters from auriferous deposits may contain up to 1 mg/L of gold. In living organisms, such as microorganisms from ore fields and marine invertebrates, gold was found at concentrations 30-750 |g/kg (Korobushkina et al. 1983). Gold has no known physiological functions and it is usually considered as inert in the elemental form, Au0 (Sadler 1976, Eisler2004). However, exposure to gold jewelry, gold dental restorations, gold implants, and beverages containing flake gold has been linked to contact allergy in susceptible individuals, mostly in the form of dermatitis. It has been proposed that dark discolorations of skin in contact with gold jewelry and dermatitis are associated with formation of the more reactive Au+ and Au3+ species due to extra- or intracellular dissolution ofAu0, e.g. by sweat or in lysosomes (Rapson 1984, Eisler 2004). Gold nanoparticles (AuNPs) - or the so-called "colloidal gold" - may be of either natural or anthropogenic origin. In the nature, AuNPs are formed by weathering of origin rocks or by mi-crobial precipitaton in auriferous soils (Southam et al. 2009). Production of anthropogenic AuNPs has been increasing due to their use in consumer, industrial and medical products. AuNPs are used for numerous applications, ranging from biosensors to catalysts, electronics, cosmetics, and cancer treatment (Unrine et al. 2010). According to the Consumer Products Inventory, there are currently 25 products (out of 1331 in total) listed containing AuNPs, mostly dietary supplements and cosmetics (Project on Emerging Nanotechnologies, 2015, retrieved on June 21st, 2016). As a result of a variety of uses, environmental release of anthropogenic AuNPs is anticipated and there is a need of further research regarding the impacts of AuNPs on the environment. At the moment, no measured environmental concentration data for AuNPs are available. However, according to Mahapatra et al. (2015), the mean annual predicted environmental concentration of AuNPs in sludge is estimated at 0.120 and 0.150 ^g/g for UK and US, respectively, and in sludge-treated soil at 300 and 150 ng/kg yearly for UK and US, respectively. Although yearly concentration is considerably low, it may increase due to continuous application over years. The bioavailability of AuNPs and their effects on terrestrial invertebrates have been studied in earthworms (Eisenia fetida; Unrine et al. 2010, 2012), fruit flies (Drosophila melanogaster; Pompa et al. 2011; Sabella et al. 2011, Vecchio et al. 2012a, 2012b) and tobacco hornworms (Manduca sexta; Judy et al. 2010, 2012). However, for terrestrial isopod crustaceans, no data on bioavailability or effects of gold ions or AuNPs exists to date. Terrestrial isopods (Porcellio scaber) are suitable model terrestrial organisms for testing effects of chemicals, since they enable precise monitoring of the exposure dose together with its consequences on different levels of biological organization (Drobne 1997). Being hyperaccumulators of various metals (Hames and Hopkin 1989), they are especially convenient in studying the biological effects of metal salts and nanoparticles (Pipan-Tkalec et al. 2010, Golobic et al. 2012, Novak et al. 2012). Skarkova et al.: Gold nanoparticles do not induce adverse effects on terrestrial isopods 35 In isopods, the most sensitive biological endpoints for studying the effects of chemicals are biochemical, histological, and physiological (Drobne 1997). These endpoints can be simultaneously investigated in the digestive glands (hepatopancreas), which are in direct contact with the substances in food. Isopod digestive glands consist of four blind-ending tubes with intestinal, hepatic and pancreatic functions. The digestive gland epithelium is built of two cell types: larger B cells with secretive and absorptive functions, which contain lipid droplets and glycogen, and smaller S cells, which predominantly accumulate metals (Hames and Hopkin 1989). After consumption of metal salt- or NP-spiked food, the digestive gland epithelium can be often found containing elevated concentrations of metal ions accumulated in storage granules (Pipan Tkalec et al. 2010, Golobic et al. 2012). Accumulation of metals in digestive glands in insoluble form is considered as a means of detoxification under exposure to elevated concentrations of metals in food (Hopkin 1990). Destabilization of the digestive gland cell membrane by NPs is a measure of cytotoxicity; however, damaged cell membrane also permits cellular internalization of NPs, which may lead to further cytotoxic effects (Novak et al. 2012). The biological endpoints at the cellular and tissue level can be combined with organism-level endpoints, such as body mass change, mortality, and avoidance behavior (Skarkova et al. 2016) to elucidate a broad picture of the effects of the tested NPs on isopods. The aim of our present study was to investigate the effects of ingested AuNPs and AuCl3 (10 and 60 |ig Au/g dry leaf) on terrestrial isopods as well as potential bioaccumulation of gold into digestive gland cells. AuCl3 was used as a positive control to account for potential dissolution of AuNPs inside the isopods' digestive tract (Eisler 2004; Golobic et al. 2012) and to differ between the effects of AuNPs and Au3+ ions. We related the data on Au bioaccumulation in digestive gland tissue to the data on the effects of Au exposure, such as mortality, weight change, feeding rate, and cell membrane destabilization of digestive gland cells. A 14-day food selection behavior test was also done to investigate their selection of the Au-spiked food. Materials and methods Test chemicals AuNPs were synthetized by the INMETRO (National Institute of Metrology, Quality and Technology; Rio De Janeiro, Brazil) as a part of the EU FP7 NanoValid project under project label NNV-004. The data on particle size, shape, Z-potential, and metal content was provided by the supplier. Gold (III) chloride (AuCl3, >99.99% trace metals basis, CAS Number 13453-07-1) and gold standard for AAS (1 mg Au/mL, TraceCERT®) were purchased from Sigma-Aldrich (Steinheim, Germany). Water used throughout the work (dH2O) was first deionized and then further purified using Elix 10/Milli-Q Gradient unit (Millipore, Bedford, Massachusetts, USA [pH = 5.7, p = 18.5 MDcm]). Physiological solution for P. scaberwas prepared according to the protocol published in Hagedorn et Ziegler (2002). Tris(hydroxymethyl)amino-methane, NaCl, KCl, MgCl2, and glucose were purchased from Merck (Darmstadt, Germany). All chemicals were of the reagent grade (EMSURE®). For the hepatopancreatic cell membrane stability assay, acridine orange solution (2% in dH2O) and ethidium bromide solution for fluorescence (~1% in dH2O) were used, both from Sigma Aldrich. Microwave acid digestion was performed with the reagent grade 65% HNO3 (Fischer Scientific, Loughborough, Leicester, UK). Test organisms Isopods P. scaber originated from the synchronized laboratory culture at the Department of Biology, University of Ljubljana, Slovenia. Cultures of P. scaber were derived from individuals collected from an unpolluted site in Polhov Gradec, Slovenia (46° 3' 0" N, 14° 18' 0" E). Animals were kept in a climate chamber at 22 ± 1 °C with a 16/8 h light/dark period (120 and 16 lx, respectively; measured using LI-1000 Data Logger, LI - COR, Nebraska, USA), caged in glass containers with moist loamy sand and peat at the bottom. They were fed with fallen leaves from various trees, with periodical additions of potatoes, fresh vegetables, and apples. 36 Acta Biologica Slovenica, 59 (1), 2016 Feeding exposure Partially decomposed common hazel leaves (Corylus avellana) were collected in the Karavanke region, Slovenia (46° 21' 32.29" N, 14° 16' 36.12" E), for the purpose of the experiment. Leaves were air-dried at room temperature (24 ± 1 °C) and stored in a cardboard box until use. Bigger leaves with minimally damaged leaf lamina were straightened and the serrated leaf edge was cut off. Leaf laminas were cut into pieces of 100 ± 10 mg. The AuNPs or AuCl3 were suspended in dH2O using a vortex (20 s, 2000 rpm) to obtain concentrations 10 and 60 ^g Au/mL. The higher concentration corresponded to the Au concentration in the original AuNP suspension provided by the supplier (0.006 % w/w), and the lower one was chosen for comparison to the results of our previous studies, e.g. Pipan-Tkalec et al. (2011). No stabilizers were used, and the chemicals were prepared freshly for each experiment. 100 ^L of Au NP dispersion or AuCl3 solution per 100 mg of leaf was applied onto the abaxial surfaces of dry leaves. This resulted in two final concentrations of 10 and 60 |ig Au/g dry leaf for both sources of Au. Control leaves were spiked with dH2O only. Spiked leaves were allowed to dry for 24 hours at room temperature. After 24 hours, dry leaves were re-weighed and this data was then used for further calculations. Only adult isopods of both sexes, and with 30-60 mg body mass were chosen for the experiments. Moulting animals (Zidar et al. 1998) and gravid females were excluded in order to keep the investigated population as homogenous as possible in terms of its physiological state. Each animal was placed individually in plastic Petri dishes (^ 9 cm), to which individual pieces of Au-treated dry leaves were added. Two experiments were carried out. The first experiment (Experiment 1) consisted of the control group and the groups exposed to AuNPs. The second experiment (Experiment 2) consisted of the control group and the groups exposed to AuCl3. In both experiments, each experimental group comprised 12 animals. The exposure conditions were the same for both experiments. Petri dishes were placed in a large, plastic-covered glass container and their humidity was maintained by periodical spraying of the internal side of the lids with dH2O. The experi- ments were maintained for 14 days in controlled and stable conditions at 22 ± 1 °C, 80% relative humidity (TFA, Dostmann GmbH et Co.KG, Wertheim, Germany), with a 16/8 h light/dark period (120 and 16 lx, respectively) and monitored on a daily basis. The food was not replaced during the exposure period, and fecal pellets were collected weekly. Post-experimental sample preparation and analysis After the 14-day exposure period, the animals were transferred to new Petri dishes and fed with uncontaminated hazel leaves for 24 h to depurate Au from their digestive system. The leaves and fecal pellets from the experiments were collected and weighed after drying at room temperature for 24 h. On the 15th day, animal mortality was recorded, and the survived animals were weighed. The experiments were considered valid if the mortality of controls did not exceed 20 % (Hornung et al. 1998). The animals were decapitated, and the hepatopancreas and gut were isolated with tweezers. One gland tube was used for the cell membrane stability assay, and other three gland tubes, the gut and the 'rest' of the body were further processed for the measurments with flame AAS. Hepatopancreatic cell membrane stability assay Cell membrane stability was tested with a modified method for the assessment of cell membrane stability, previously described by Valant et al. (2009). A single isolated hepatopancreatic tube was incubated for 5 minutes in a mixture of the fluorescent dyes acridine orange and ethidium bromide and then put on a microscope slide. Fresh samples were photographed and examined by the Axioimager.Z1 fluorescent microscope (Carl Zeiss, Jena, Germany) with two different sets of filters. The excitation filter of 450 to 490 nm and the emission filter of 515 nm were used to visualize acridine orange- and ethidium bromide-stained nuclei, and the excitation filter of 365 nm and the emission filter of 397 nm were used to visualize nuclei stained with ethidium bromide only. Cell membrane integrity was assessed by visual examination of micrographs and classified Skarkova et al.: Gold nanoparticles do not induce adverse effects on terrestrial isopods 37 from 1 to 10 according to a predefined scale. On the basis of preliminary experiments, the control animals showing less than 10 % of nuclei stained by ethidium bromide were classified as 1 or 2. The animals exposed to AuNPs, AuCl3, but showing less than 10 % of nuclei stained by ethidium bromide, were also classified as 1 or 2 (Valant et al., 2009). Gold content determination Each isopod body part was placed on a separate small piece of a filter paper (approximately 4 mm*7 mm size) and stored in a plastic tube. Prior to analysis, samples were acid digested in concentrated HNO3 in the Milestone Ethos E (Bergamo, Italy) microwave lab station equipped with SK-10 high-pressure segmented rotor and 3 mL quartz microsampling inserts. Digestion was conducted at 180°C and 600 W power, with step 1 (heating) lasting 15 min, step 2 (constant temperature) lasting 10 min, and 45 min cooling to 60°C. Total Au concentrations in the three parts of each animal (one digestive gland, the gut and the 'rest' of the body) were measured by flame AAS (Perkin-Elmer AAnalyst 100, Waltham, Massachusetts, USA). Metal spiking recovery was determined by measuring the Au concentrations on the remnants of leaves after the experiment. Food selection behavior test The food selection behavior test (Experiment 3) was carried out according to the protocol by Zidar et al. (2004). The preparation of spiked food and the selection of animals were conducted in the same way as in the toxicity endpoint tests. Particular attention was paid to include only the animals with intact antennae. Each of the five experimental groups included 15 animals, which were caged individually in plastic Petri dishes (^ 9 cm) and offered two hazelnut leaf pieces (approximately 100 mg each) of different shape (square and triangular) for 14 days. The two leaf pieces had been treated differently. The first piece was control (spiked with dH2O only) and the second one was spiked with a test compound. The tested Au sources and concentrations were the same as in the toxicity endpoint tests, i.e. AuCl3 and AuNPs at concentrations 10 and 60 ^g Au/g dry leaf. As a positive control, CoCl2*6H2O with the nominal exposure concentration 2000 ^g Co2+/g dry leaf was used. In our previous work (data not published), isopods P. scaber significantly avoided Co2+ contaminated food at the same nominal exposure concentration, therefore we used only 10 animals in this group for ethical reasons. This collectively resulted in 6 combinations of test compounds: "0 (control) vs. 0", "+control (Co2+) vs. 0", "AuNPs 10 vs. 0", "AuNPs 60 vs. 0", "AuCl3 10 vs 0" and "AuCl3 60 vs. 0". The experimental conditions and post-experimental procedure were the same as in the toxicity endpoint tests, except that animal dissection and tissue processing were not performed. The total food consumption rate was calculated as the amount of food consumed (both leaves offered) during 14 days, per fresh animal weight. The consumption rate of a single leaf was calculated as the percentage of the total food consumption rate. This data was then used for the calculation of food selection response. Dead animals were excluded from the calculations. Data analysis In all experiments, 12 or 15 animals per each tested group were exposed, but the number of analyzed animals after the experiments was lower due to mortality caused by moulting and due to development of marsupia in females; all such animals were excluded from further data processing. The numbers of analyzed animals are presented in the Figures as part of the x-axis labels. Data is presented as mean values, and uncertainties are expressed as standard deviations (SD). Average animal fresh body mass (per individual) during the experiment was calculated as an arithmetic average of fresh body masses recorded before and after the experiment. Feeding rate (per individual) was calculated by divinding the total mass of consumed leaves during the experiment with the average animal fresh body mass. Since the animals collected for Experiments 1 and 2 belonged to the same population, the corresponding control groups were tested for homogeneity of variances with the Fliegner-Killeen test and their average body masses and feeding rates were compared by the Mann-Whitney C-test using R statistical package (R Development Core Team 2015). Because no significant differences were found (p>0.05), the controls were pooled before 38 Acta Biologica Slovenica, 59 (1), 2016 further data analysis. Statistical significance of differences between the pooled control and the animals exposed to Au compounds was assessed by the Mann-Whitney Latest using OriginPro 8.0 software (OriginLab, Northampton, MA, USA). Results Nanoparticle characteristics Gold NPs were received from the INMETRO as a reddish dispersion in water. As stated by the manufacturer's specifications, the Au NP dispersion contained 0.006 % (w/w) of Au, the nominal average particle size was 15.7 nm, and Z-potential was -30.4 mV. The shape of nanoparticles was spheroidal (Fig. 1). Figure 1. Transmission electron micrograph of Au nanoparticles; the photograph was provided by the INMETRO (National Institute of Metrology, Quality and Technology; Rio de Janeiro, Brazil). Slika 1. Presevna elektronska mikrografija nan-odelcev Au; sliko je posnel INMETRO (Nacionalni inštitut za metrologijo, kakovost in tehnologijo; Rio de Janeiro, Brazilija). Mortality, body mass change and food consumption of isopods Fourteen days of exposure to Au compounds had no statistically significant effect on mortality, average body mass (Fig. 2a) or feeding rate (Fig. 2b) of the test animals in comparison to the control in any of the exposure groups (Mann-Whitney L-test, p>0.05, not marked on Fig. 2). Digestive gland cell membrane stability Valant et al. (2009) demonstrated that the digestive gland cell membrane stability value was rarely higher than 2 in the animals from the stock culture, which are in good physiological condition, and this was taken as a benchmark. The higher the value, the more the membrane is destabilized, and the cell membranes are considered completely destabilized when the value is 10. In the present study, in the animals from the stock culture, in control animals and in those exposed to Au-spiked food, the values were never higher than 2, which indicates that neither AuCl3 nor AuNPs affected membrane stability of the digestive gland cells (data not shown). Gold content in food and animal tissues The measured metal concentrations on spiked leaves were within 10 % of the nominal values for both tested Au sources at both tested concentrations. No Au was found in the hepatopancreas, the gut or the rest of the body of the animals from any of the exposure regimens, regardless of the Au source or concentration (data not shown). Skarkova et al.: Gold nanoparticles do not induce adverse effects on terrestrial isopods 39 Figure 2. Average body mass (A) and feeding rates (B) of P. scaber isopods during 14-day exposure to Au-spiked food. The animals were fed non-spiked food (control) or food that was spiked with AuCl3 salts (AuCl3 10 and AuCl3 60, per nominal Au concentrations) or Au nanoparticles (AuNPs 10 and AuNPs 60, per nominal Au concentrations). The controls from Experiment 1 (with AuNPs) and Experiment 2 (with AuCl3) were pooled because no significant differences between them were found in any of the tested parameters. The nominal exposure concentrations of Au are provided on the x-axis. The symbols on the box plot represent maximum and minimum values (whiskers: mean values (■), outliers (—); n = number of specimens in each test group. Slika 2. Povprečna telesna masa (A) in stopnja prehranjevanja (B) enakonožcev P. scaber med 14-dnevno izpostavitvijo hrani, tretirani z Au. Živali smo hranili z netretirano hrano (kontrola) ali hrano, na katero smo nanesli AuCl3 (AuCl3 10 in AuCl3 60, za nominalne koncentracije Au) oziroma nanodelce Au (AuNPs 10 in AuNPs 60, za nominalne koncentracije Au). Kontrolni skupini iz poskusa 1 (z nanodelci Au) in poskusa 2 (z AuCl3) smo združili, saj med njima ni bilo statistično značilnih razlik v nobenem izmed testiranih parametrov. Nominalne izpostavitvene koncentracije Au so navedene na x-osi. Simboli na okvirjih z ročaji predstavljajo minimalne in maksimalne vrednosti distribucije (ročaji: povprečja (■) in osamelce (—); n = število osebkov v vsaki testni skupini. 40 Acta Biologica Slovenica, 59 (1), 2016 Food selection behavior test In the Experiment 3, avoidance or preference behavior was not demonstrated (Fig. 3). The masses of consumed leaves did not significantly differ (Mann-Whitney Latest, p>0.05) between the two leaf pieces (control and Au-spiked) for both tested Au compounds (AuNPs and AuCl3) and at both exposure concentrations (10 and 60 ^g Au/g dry food). Significant preference for non-spiked over CoCl2-spiked leaves was found in the positive control group (Fig. 3; Mann-Whitney L-test, p<0.001). Figure 3. Food selection of P. scaber isopods. Animals were exposed to untreated food (0) and food treated with AuNP or AuCl3 simultaneously for 14 days. The nominal exposure concentrations of Au are provided on the x-axis (in ^g Au/g dry leaf). CoCl2x6H2O with the nominal exposure concentration 2000 ^g Co2+/g dry leaf was used as a positive control (+control). The food choice (%) is presented as the ratio between consumption rates of the two offered leaves, expressed as mean percentages of the total food consumption rate for each exposure group. Uncertainties are expressed as standard deviation (SD). Significant differences: p<0.001 (***); n = number of specimens in each test group. Slika 3. Izbira hrane pri P. scaber. Živali so bile 14 dni hkrati izpostavljene netretirani hrani (0) and in hrani, na katero smo nanesli AuCl3 oziroma nanodelce Au. Nominalne izpostavitvene koncentracije Au so navedene na x-osi (v ^g Au/g suhe mase lista). Kot pozitivno kontrolo (+control) smo uporabili CoCl2x6H2O v nominalni izpostavitveni koncentraciji 2000 ^g Co2+/g suhe mase lista. Izbira hrane (%) je predstavljena kot razmerje med stopnjama prehranjevanja z dvema ponujenima listoma ter izražena kot povprečna vrednost stopnje prehranjevanja za vsako testno skupino posebej. Negotovost je izražena s standardnimi deviacijami (SD). Statistično značilne razlike: p<0.001 (***); n = število osebkov v vsaki testni skupini. Skarkova et al.: Gold nanoparticles do not induce adverse effects on terrestrial isopods 41 Discussion In the present study we assesed the influence ofAuNPs and AuCl3 on different biological endpoints in the model organisms, terrestrial isopods P. scaber. We tested the adverse biological effects of AuNPs and AuCl3 as detectable by common toxicoloxical parameters (body mass change, food consumption rate, metal feeding preference/avoidance behavior, and mortality) and cell membrane integrity assay, which provides information onf the cell membrane destabilization. In parallel, we assessed the Au3+ assimilation in digestive glands. Our results show that AuNPs did not affect P. scaber mortality, body mass (Fig. 2a) or food consumption (Fig 2b) when exposed through food at concentrations 10 and 60 ¡ig Au/g for 14 days. In other words, we showed that AuNPs and AuCl3 do not affect the organism-level endpoints in P. scaber at relatively low exposure concentrations. Our results are in line with previous reports on earthworms (Unrine et al. 2010). In E. fetida, exposure to AuNPs (20 and 55 nm, 5 ¡g Au/g dry mass spheres) in soil for 28 days did not affect their mortality or growth (Unrine et al. 2010). In contrast, feeding with 15 nm citrate-capped AuNPs at concentrations 3 and 27 ^g AuNPs/g food per day reduced the lifespan of D. melanogaster for 24 and 41 %, respectively, in comparison to control (average control population half-life was 80 days; Sabella et al. 2011). In the same experimental setup and with the same AuNPs, the lifespan was reduced for 62 % when D. melanogaster were exposed to 12 |ig AuNPs/g food per day (average control population half-life was 37 days; Pompa et al. 2011). However, the exposure doses for D. melanogaster (Pompa et al. 2011, Sabella et al. 2011) were higher than in our study (0.7 and 4.3 ^g Au/g food per day for nominal exposure concentrations 10 and 60 ^g Au/g dry leaf, respectively), which may partially explain why mortality in P. scaber in our study was not elevated in comparison to D. melanogaster. In line with this, we also showed that isopods show no preference/avoidance towards Au-spiked hazelnut leaves for both tested Au compounds (AuCl3 and AuNPs; Fig. 3). This is in agreement with the observation that feeding rate of isopods was unaffected upon Au exposure (Fig. 2b). Avoidance behavior of isopods towards metal- contaminated food has previously been shown (Zidar et al. 2004; Skarkova et al. 2016). Nanoparticles may pass from the gut into the lumen of the P. scaber hepatopancreas during digestion, as it has been demonstrated for TiO2 NPs (Novak et al. 2012) and WOX nanotubes (Novak et al. 2013), and cause cell membrane destabilization (Novak et al. 2012, 2013). However, neitherAuNPs norAuCl3 caused the reduced cell membrane integrity of hepatopancreatic cells in our current study. The results of cell membrane integrity assay match the data on the Au assimilation. Namely, neither of the P. scaber body parts contained any gold, which indicates that AuNPs were not internalized into tissues and Au3+ ions were not assimilated into the digestive gland cells. In general, we cannot exclude the possibility that P. scaber possesses the assimilation capacity for Au3+, because Au has the affinity for sulphur-bearing ligands (Ko-robushkina et al. 1983), which are present in the type B granules of the hepatopancreatic S cells (Hopkin 1990) and enable the assimilation of other metals with affinity for sulphur, such as cadmium, copper, lead and mercury (Hopkin 1990), silver (Pipan-Tkalec et al. 2011) and cobalt (Novak et al. 2013). Nor we can exclude the possibility for the dissolution of AuNPs inside the P. scaber digestive tract (Golobic et al. 2012), since the dissolution of gold can be induced by biological macromolecules, most notably amino acids and proteins, as well as bacteria (Sadler 1976, Rapson 1982, Korobushkina et al. 1983). However, under employed exposure conditions, the internalization/ assimilation ofAu into P. scaber digestive glands clearly did not occur. Our results contrast those for E. fetida (Un-rine et al. 2010), M. sexta caterpillars (Judy et al. 2010, 2012), and D. melanogaster (Pompa et al. 2011) where AuNPs, administered via food, were found in tissues surrounding the digestive tract (including the digestive tract epithelium in E. fetida) as well as in the reproductive organs in D. melanogaster. The presence and localization of gold was corroborated by transmission electron microscopy (TEM) coupled to energy dispersive spectroscopy ([EDS] Unrine et al., 2010), X-ray absorption near edge spectroscopy (^XANES) and synchrotron X-ray fluorescence microprobe ([^XRF]; Judy et al. 2010, 2012) or high angle annular dark field scanning transmission electron 42 Acta Biologica Slovenica, 59 (1), 2016 microscopy ([HAADF-STEM]; Pompa et al. 2011). No evidence of dissolution ofAuNPs was observed in the quoted studies, so NPs were likely assimilated intact (Unrine et al. 2010, Judy et al. 2010, Pompa et al. 2011). In conclusion, our results denote that concentrations of Au salt and AuNPs tested in this work (10 and 60 ^g Au/g dry leaf) do not induce adverse effects on terrestrial isopods after 14 days of exposure. These concentrations are higher than currently predicted environmental levels (Mahapatra et al. 2015), which further confirms the finding that AuNPs are safe for isopods. In general, AuNPs are among NPs with the least toxic potential for test organisms commonly employed in environmental studies. Namely, Bondarenko et al. (2016) have used a set of assays to screen seven NMs using 14 different test species and cell lines. The toxicity decreased in the following order: Ag > ZnO > CuO > TiO2 > MWCNTs > SiO2 > Au (Bondarenko et al. 2016). According to our perception, the majority of authors in nanotoxicity research encourage the publication of those results that show some kind of effects of nanoparticles on organisms. We therefore suggest that also those data with no documented effects should be published in equal proportion to help built a realistic public perception of the nanomaterial environmental risk. Acknowledgements The paper is part of the PhD work of Pavlina Skarkova, working under the supervision of Professor Milada Vavrova. PS received ERASMUS trainee scholarship for 5-month study visit in Slovenia under Grant Number ERA-M0B-2013-25. The investigation was supported by the research programs and projects financed by the Slovenian Research Agency (P1-0184, J1-4109) and within the FP7 EU Project "NANOVALID" (Contract No. 263147). TR acknowledges the grant "Innovative scheme of co-funding doctoral studies for promoting co-operation with the economy and solving of contemporary social challenges" under Grant Number 1291, received from Ministry of Education, Science, and Sports of Republic of Slovenia. Povzetek Nanodelci zlata se čedalje pogosteje uporabljajo za različne aplikacije, kot je npr. v kozmetiki, barvah in elektrokemijskih senzorjih ter v detekciji, diagnostiki in zdravljenju tumorjev. Kljub temu še vedno ni dovolj podatkov o njihovih potencialnih negativnih učinkih na kopenske organizme. V tej študiji smo proučevali vpliv okoljsko relevantnih koncentracij ND Au na kopenske rake vrste Porcellio scaber po 14-dnevni izpostavitvi. Živali smo izpostavili listom, na katere so bili nanešeni nanodelci zlata. Proučevali smo vpliv na smrtnost, maso živali, hitrost prehranjevanja, izogibalno vedenje in destabilizacijo membrane celic prebavnih žlez. Izmerili smo tudi asimilacijo Au v prebavnih žlezah. Rezultati so pokazali, da nobena izmed testiranih koncentracij ND Au (10 in 60 |ig Au/g lista) po 14-dnevnem hranjenju ni imela vpliva na kopenske rake. Prav tako nismo izmerili prisotnosti Au v prebavnih žlezah, kar kaže, da se ND Au niso niti raztapljali v prebavnem soku niti vstopali v celice prebavnih žlez. Enake rezultate smo dobili tudi z referenčno kovino, soljo AuCl3. Zaključujemo, da so testirane koncentracije ND Au varne za kopenske rake. Naše mnenje je, da se na področju nanotoksikologije trenutno objavlja predvsem rezultate, ki kakorkoli nakazujejo na negativni vpliv nanodelcev na organizme. Zato želimo stimulirati tudi objavo rezultatov, ki kažejo nasprotno. Na ta način bomo pripomogli k bolj uravnoteženemu ter realističnemu mnenjujavnosti o okoljski varnosti nanomaterialov. Skarkova et al.: Gold nanoparticles do not induce adverse effects on terrestrial isopods 43 References Bondarenko, O. M., Heinlaan, M., Sihtmae, M., Ivask, A., Kurvet, I., Jemec, A., Mannerstrom, M., Heinonen, T., Rekulapelly, R., Singh, S., Zou, J., Pyykko, I., Drobne, D., Kahru, A., 2016. 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ACTA BIOLOGICA SLOVENICA LJUBLJANA 2016 Vol. 59, Št. 1: 45-55 Am No chronic effects on biochemical biomarkers, feeding and survival of carnolian honeybees (Apis mellifera carnica) after exposure to nanosized carbon black and titanium dioxide Kronična izpostavitev nanomaterialom titanovega dioksida in črnemu ogljiku nima vpliva na biokemijske biomarkerje, prehranjevanje in preživetje kranjske čebele (Apis mellifera carnica) Anita Jemec, Tamara Milivojevic, Damjana Drobne, Kristina Sepčic, Janko Božič, Gordana Glavan* University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, Ljubljana SI-1000, Slovenia Correspondence: gordana.glavan@bf.uni-lj.si Abstract: Honeybees (Apis mellifera) are important pollinators threatened by environmental pollution, plan protection products and other potential contaminants. Due to an extensive predicted use of engineered nanomaterials (NMs) in agriculture the impact on honeybees should be investigated. We studied the 10-days chronic dietary effect of carbon black (CB) and titanium dioxide (TiO2) NMs on the antio-xidant activities, cholinergic function, feeding behaviour and survival of honeybees. Exposure of honeybees Apis mellifera carnica to TiO2 and CB NMs (1 mg ml-1) did not affect the feeding and survival. No alteration of catalase, acetylcholinesterase and glutathione S-transferase enzymatic activity was noticed in the brain of honeybees, indicating that TiO2 and CB NMs at the tested exposure dose had no adverse effects on honeybees. Currently predicted environmental concentrations for TiO2 and CB NMs are significantly lower than the concentration tested in the current study. Based on our findings we conclude that the potential use of TiO2 and CB NMs in agriculture is currently safe for honeybees at the tested concentration level and presents potential advantages compared to other NMs with known toxic potential. Keywords: nanopesticide, carbon black nanomaterial, titanium dioxide nano-material, acetylcholinesterase, glutathione S-transferase, catalase, feeding behaviour. Izvleček: Medonosne čebele (Apis mellifera) so pomembni opraševalci, ogroženi zaradi onesnaževanja okolja, fitofarmacevtskih sredstev in drugi možnih onesnaževalcev. Zaradi široke predvidene uporabe inženirsko proizvedenih nano-materialov v poljedelstvu je potrebno raziskati njihov vpliv na medonosne čebele. V tej študiji smo preučili 10-dnevni kronični prehranski učinek nano-črnega ogljika (nČO) in nano-titanovega dioksida (nTiO2) na prehranjevalno vedenje, antioksidativno aktivnost, na delovanje holinergičnega živčnega sistema in preživetje čebel. Pokazali smo, da nTiO2 (1 mg ml-1) in nČO (1 mg ml-1) nista vplivala na hranjenje in preživetje kranjskih čebel Apis mellifera carnica. Hranjenje z obema vrstama nanomaterialov 46 Acta Biologica Slovenica, 59 (1), 2016 ni vplivalo na aktivnost treh biomarkerskih encimov katalaze, acetilholinesteraze in glutation S-transferaze v možganih čebel, kar pomeni, da nanomateriali verjetno niso imeli škodljivega učinka na čebele. Trenutno ocenjene napovedane vrednosti okoljskih koncentracij za nTiO2 in nČO so znatno nižje od teh, ki smo jih uporabili v sedanji študiji. Na podlagi naših ugotovitev sklepamo, da je morebitna uporaba nTiO2 in nČO NM v kmetijstvu varna za čebele v okviru testiranih koncentracij in predstavlja potencialno prednost v primerjavi z nanomateriali z znanim toksičnim potencialom. Ključne besede: nanopesticidi, nano-črni ogljik, nano-titanov dioksid, acetilho-linesteraza, glutation S-transferaza, katalaza, prehranjevalno vedenje. Introduction Honeybees (Apis mellifera) are important pollinators and many agricultural crops depend on pollination. Colony collapse disorder (CCD) causes massive deaths in honeybees and significant economic losses (van Engelsdorp et al. 2009). In the recent years it is evident that CCD is caused by the combined action of parasites, pathogens and pesticide stressors (Sánchez-Bayo et al. 2016). Synergistic interactions among the parasitic mite Varroa, viral pathogens and pesticides could severely reduce host immune competence potentiating the sensitivity of honeybees to other possible stress agents. Among the stress agents, pesticides are now widely studied while other emerging environmental contaminants, such as nanomaterials (NM) are still highly neglected. Engineered nanomaterials are extensively used in agriculture to reduce amount of applied plant protection products, minimize nutrient losses in fertilization, and increase yields through an optimized nutrient management (Gogos et al. 2012; Kah and Hofmann 2014). New formulations containing nanomaterials are called "nanopesticides" and "nanofertilisers" (Kah and Hofmann 2014). Up to 3000 of patents and over 100 peer-reviewed publications directly related to nanopesticides have been published until 2011 (Kah and Hofmann 2014), indicating intense research activity in this field. Nano-sized titanium dioxide (TiO2 NM) was one of the first nanomaterials commercially available and is used in a wide variety of materials and applications, including self-cleaning surface coatings, light-emitting diodes, solar cells, disinfectant sprays, sporting goods, water-treatment agents, cosmetics and agriculture (IARC/WHO, 2010). These NMs are widely used due to their high stability, anticorrosive and photocatalytic properties. In agriculture, TiO2 NM is added to pesticide formulation to catalyse the photodegradation of the pesticide organic active ingredient. Examples of such nanoformulations with TiO 2 are: chlorfenapyr (Cao et al. 2005), imidacloprid and avermectin (Guan et al. 2008, 2011). TiO2 NMs are also interesting in terms of their antimicrobial activity and several studies suggest their suppressing action on bacterial and fungal pathogens growth on crops (Paret et al. 2013 a,b). Until 2013 70 patents including TiO2 have been registered as nanopesticides (Kah et al. 2013). Nano-sized carbon black (CB NM) is produced at rates of several million tons per year (Navarro et al. 2008). It is a product of incomplete combustion of fossil fuels and vegetation, and is used in rubber production, as black pigment in printing inks, as electrodes in batteries, and in leather production. In comparison to TiO2 NM, CB NM is much less explored in terms of potential hazard for the environment. Significantly more information is available for other carbon nanomaterials, such as carbon nanotubes and fullerenes (Jackson et al. 2013). Insects, including honeybees, are among the least investigated non-target organisms in terms of potential nanomaterial hazard (Garner et al. 2015). Currently only one study on the effect of NM (zinc oxide; ZnO) on honeybees exists (Milivojevic et al. 2015). Honeybees (Apis mellifera) are potentially exposed to TiO2 and CB NM due to their foraging behaviour via contact with contaminated plants as well as water droplets. Flying bees, beehives or flowers attracting the bees may come into contact with NM also via traffic dust contaminated with TiO2 and CB NM (Perez et al. 2010). Jemec et al.: Effects on honeybees after exposure to nanosized carbon black and titanium dioxide 47 In the present work, we aimed to investigate the chronic effects of CB and TiO2 NM on feeding behaviour, survival and stress enzyme activities in the honeybee brain. Three commonly applied biomarkers of exposure and effect were measured: antioxidant enzymes catalase (CAT) and glutathione S-transferase (GST), and neurotoxic biomarker acetylcholinesterase (AChE) (Jemec et al. 2010). Catalase decomposes hydrogen peroxide (Halliwell and Gutteridge 2007). GSTs are a family of detoxification enzymes, which catalyse the conjugation of glutathione with xenobiotics and cytotoxic aldehydes produced during lipid peroxidation. GSTs are considered as both antioxidant and detoxification enzymes (Barata et al. 2005). Acetylcholinesterase plays an established role in cholinergic transmission by hydrolysing the neurotransmitter acetylcholine thereby terminating the synaptic transmission (Kim and Lee 2013). In addition, a number of non-neuronal functions of AChE have also been proposed (Karczmar 2010). A disruption of the honeybees' neuronal cholinergic signalling affects their orientation, olfactory learning and navigation abilities, which results in their failure to return to hives, even potentially leading to CCD (Farooqui 2013). Knowledge on the effect of pollutants on the cholinergic system of honeybee brain is therefore important for understanding the potential environmental hazard of NMs. Materials and methods Chemicals The following chemicals were purchased from SigmaAldrich (Germany): monobasic and dibasic potassium phosphate, 1-chloro-2,4-dinitrobenzene, L-glutathione (reduced form), 5,5'-dithiobis-2-nitrobenzoic acid, sodium hydrogen carbonate, acetylthiocholine chloride, sodium sulphate and ethylenediaminetetraacetic acid. BCA Protein Assay Reagent A and Reagent B were purchased from Pierce (US). All chemicals were of the highest commercially available grade, typically >99%. CB nanopowder was provided by PlasmaChem GmbH (Berlin, Germany) and TiO2 NM was provided by Nanologica (Sweden) in the framework of the EU FP7 NanoValid project. Preparation and characterisation of NM suspensions A suspension of TiO2 NM or CB NM was prepared by adding nano powder to 1.5 M sucrose solution in milli-Q water with sonication (PIO Iskra, Sonnys 2GT; 40 kHz, 2x100 W) of the suspension for 24 h. For better comparison with the effects of ZnO NM on honeybees, published by Milivojevic et al. 2015, where the same experimental set-up and tested concentrations were applied, we used final nominal concentration 1 mg mL-1 of NMs in sucrose. The properties of CB NM were as reported by Mesaric et al. (2013). CB is composed of amorphous, globular primary nanoparticles with diameter of about 20 nm (Supplementary information Fig.S1A). The primary sizes of TiO2 NM were in range between 110 and 170 nm, showing a large size distribution (Supplementary information Fig.S1B). The secondary size of NMs in the 1.5 M sucrose solution was not measured since the size of NM aggregates/agglomerates alters in different fluids present in the honeybee digestive system. Therefore, it is important to bear in mind that the data on the size of aggregates in the sucrose solution have no actual correlation with the actual size of NM in the digestive system of honeybees. Test animals Adult summer honeybee workers (Apis mel-lifera carnica, Pollman 1879) were randomly collected inside the hive from colonies that were maintained according to the good beekeeping practice at the Biotechnical Faculty, University of Ljubljana, Slovenia. Honeybees were then transferred to wooden cages (9.5 x 4 x 7.5 cm) and supplied ad libitum with water and 1.5 M sucrose solution in gravity feeders until all bees were collected. All bees were maintained in cages for 1 h at 27 °C before treatments. Honeybee exposure to TiO2 and CB NM via food A group of 120 bees was divided into 6 groups of 20 animals. Each group of bees (20 specimens) was placed into separate wooden cage. Two cages with a control group were fed with 1.5 M sucrose 48 Acta Biologica Slovenica, 59 (1), 2016 solution only, two groups of bees received a suspension of TiO2 NM in 1.5 M sucrose (1 mg mL-1 TiO2) and two groups were fed with a suspension of CB NM in 1.5 M sucrose (1 mg mL-1 CB). Each cage received a syringe with tap water. The sucrose solution and water were renewed every 2 days. The cages were placed for 10 days in an incubator at 27 °C and 95% relative humidity during the overall exposure period. The feeding was estimated as the volume of total consumed solutions/suspensions per exposure group after 10 days. Mortality of bees was monitored during 10 days of the experiment. Brain dissection and homogenization After 10-days of dietary exposure to NM, the brains were isolated from honeybees according to Carreck et al. (2013). Isolated brains were submersed into a droplet of honeybee Ringer solution, both hypopharyngeal and postcerebral glands were removed. The brains were then stored at -20 °C until analysis. Individual brains were homogenized in 200 mL of 100 mM potassium phosphate buffer (pH 7.4) per sample. Individual homogenates were centrifuged for 15 min at 12,092 g and 4 °C, and the supernatants were stored at -20 °C for enzyme analyse. Enzyme and protein assays AChE, GST and CAT activity measurements AChE activity was analysed according to Ellman et al. (1961), GST activity was analysed according to the method of Habig et al. (1974) and CAT activity was determined according to Jemec et al. (2008). All procedures were adapted as described in Milivojevic et al. (2015) and Jemec et al. (2008). AChE activity was expressed in nmoles of hydrolyzed acetylthiocholine chloride/ min/mg protein (extinction coefficient s405=13,600 M-1 cm-1). GST activity was expressed in nmoles of conjugated GSH/min/mg protein (extinction coefficient s340=9600 M-1 cm-1). The CAT activity was expressed as ^moles of degraded hydrogen peroxide/min/mg protein (extinction coefficient, e240 = 43.6 M-1 cm-1). Protein quantification Protein concentration in the supernatant of honeybee brain homogenates was analyzed using a BCA™ Protein Assay Kit, a modification of the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Statistical analysis The significant differences between the control and exposed groups of animals were determined by Kruskal-Wallis analysis and Mann-Whitney U test (p<0.05) using OriginPro software. Results Feeding and survival After 10-days of exposure to CB NM the total volume of consumed suspensions of CB NM (1 mg mL-1) in 2 groups of 40 individuals was similar to the total volume of consumed sucrose solution in 2 groups of 40 individuals indicating that CB NM did not affect the feeding when comparing treated and control groups of honeybees (Fig.1). In honeybees (2 groups, n=40) exposed to the TiO2 NM (1 mg mL-1), we observed a slightly (13.9 %) higher food uptake than in honeybees fed with control sucrose solution (Fig.1), but this increase was not statistically significant. The total volume of consumed suspensions of CB NM, TiO2 NM and control sucrose solution in 10-days were 15.8 ml (8 mL and 7.8 mL), 18 mL (9 mL and 9 mL) and 16 mL (8 mL and 8 mL) per treatment (and per groups), respectively (Fig.1). The chronic 10-days oral exposure of honeybees to TiO2 (1 mg mL4) and CB NM (1 mg mL-1) did not affect the survival in both treated groups (data not shown). There was no morality in all groups during the 10-days exposure period. Enzyme activities after exposure to tested substances Chronic 10-days exposure to TiO2 NM (1 mg mL-1) or CB NM (1 mg mL-1) did not alter significantly the activities of brain AChE, CAT and GST (Fig.2) (Kruskal-Wallis analysis and Jemec et al.: Effects on honeybees after exposure to nanosized carbon black and titanium dioxide 49 Figurel: The effect of titanium dioxide (TiO2 NM) and nano carbon black (CB NM) on honeybee feeding. Total volume of consumed food (mL) in bees after 10-days exposure to TiO2 NMs suspension (1 mg mL-1) and to CB NM suspension (1 mg mL-1) is shown (N=40). Slika 1: Vpliv nano-črnega ogljika (nČO) in nano-titanovega dioksida (nTiO2) na prehranjevanje medonosne čebele. Prikazana je skupna količina porabljene hrane (mL) pri čebelah po 10-dnevni izpostavljenosti suspenziji nTiO2 (1 mg mL-1) ali nČO (1 mg mL-1) (N = 40). Mann-Whitney U test (p<0.05)). The mean (±SE) activity of AChE in bee brains (Fig. 2A) after chronic oral exposure to TiO2 NM and CB NM were 2.08±0.16 and 2.08± 0.16 nmol/min/mg protein, respectively, and were similar to control values (1.91±0.15 nmol/min/mg protein). The mean activity of GST in bee brains (Fig. 2B) after chronic oral exposure to TiO2 NM and CB NM were 4.15±0.13 and 4.81± 0.12 nmol/min/ mg protein, respectively, and were similar to the control values (4.59±0.15 nmol/min/mg protein). The mean activities of CAT in bee brains (Fig. 2C) after chronic oral exposure to TiO2 NMs and CB NMs were 0.64 ± 0.046 and 0.77 ± 0.044 ^mol/ min/mg protein, respectively, and were similar to control values (0.72 ± 0.048 |imol min/mg protein). Discussion The results of the present study show that the TiO2 (1 mg mL-1) and CB NMs (1 mg mL-1) do not cause any adverse effects, neither sub-lethal nor lethal, on honeybees Apis mellifera carnica after a 10-days chronic dietary exposure. No effects on survival, feeding behaviour, antioxidant activities (CAT and GST), and cholinergic enzyme activity (AChE) in the brain of honeybees was observed. We have previously anticipated that TiO2 NM and CB NM might not be highly toxic to honeybees, since these two materials have been previously recognised as presumably inert, e.g. having little biological interaction with the test organisms (Bondarenko et al. 2016). Namely, Bondarenko et al. (2016) have used a set of assays to screen seven NMs using 14 different test species and cell lines. The toxicity decreased in the following order: Ag>ZnO>CuO>carbon nanotubes>Au>SiO2=TiO2. It is now well established that the toxicity of the NMs is mainly driven by two basic intrinsic properties of NMs: high solubility (e.g. Ag, CuO and ZnO) and high aspect ratio (describes the proportional relationship between its diameter and its length) (e.g. carbon have high aspect ratio). TiO2 and CB NMs are neither soluble in aqueous media nor have high aspect ratio, which could explain their low toxic potential. In contrast to soluble NMs, both TiOrand CB NMs have high adsorption potential for the body 50 Acta Biologica Slovenica, 59 (1), 2016 Figure 2: Acetylcholinesterase(A), glutathione-S-transferase (B), and catalase (C) activity in honeybee brains after chronic exposure to TiO2 NM (1 mg mL-1) and CB NMs (1 mg mL-1) (N=40). Slika 2: Aktivnost acetilholinesteraze (A), glutation S-transferaze (B) in katalaze v možganih čebel po kronični izpostavitvi nTiO2 (1 mg mL-1) ali nCO (1 mg mL-1) (N=40). Jemec et al.: Effects on honeybees after exposure to nanosized carbon black and titanium dioxide 51 surface which was shown to induce adverse effects on organisms (Xia et al. 2011; Mesaric et al. 2015a, b; Nielsen et al. 2008). CB NM adsorbed on the sperm and embryo of brown algae Fucus serratus, to body surface of A. salina larvae (Mesaric et al. 2015a) and to sperm of sea urchin (Paracentrotus lividus) (Mesaric et al. 2015b), resulted in abnormal development of algae, inhibition of larvae swimming, and reduced fertilisation of sea urchin eggs, respectively. The adsorption of TiO2 NM on crustaceans Daphnia magna (Dabrunz et al. 2011) and algae (Aruoja et al. 2009) had a negative effect on the immobility and growth, respectively. Based on available literature data on TiO2 and CB NM and their high adsorption potential these NM could adsorb onto the digestive tract surface of honeybees, which may result in feeding disruption. However, this potential effect was not confirmed during 10-days exposure of honeybees to 1 mg mL-1 of CB and TiO2 NMs. Chronic exposure of honeybees to CB and TiO2 NMs did not significantly change the feeding, but we observed a slightly higher food uptake in honeybees fed with TiO2 NM in comparison to honeybees that received only control sucrose solution. Behavioural response such as feeding alteration due to possible stress agents could occur before the alterations in biochemical biomarkers (Hellou 2011). Currently, no data in the literature are available confirming that honeybees are able to sense metals. The effect of different metals on honeybees behaviour was explored only by few studies (Hladun et al. 2012; Burden et al. 2016; S0vik et al. 2015), and only in our last study the effects of metallic NMs was addressed (Milivojevic et al. 2015). In our study with tested ZnO NMs we found that chronic exposure did not alter the feeding in honeybees whereas Zn2+ salt increased the feeding (Milivojevic et al. 2015). Due to potential environmental burden of CB and TiO2 NMs it would be important to investigate the possible preference/avoidance towards solutions contaminated with these NMs. Catalase, GST and AChE are among the most commonly applied biochemical biomarkers of toxicant-induced physiological changes in organisms (Jemec et al. 2010). It has been previously shown that 1 mg mL-1 of CB NM caused an increase of GST and AChE activities in Artemia salina after 48 h exposure (Mesaric et al. 2015a), while no alteration of CAT was reported. CB or TiO2 NMs (0.005 mg mL-1) increased the activities of CAT and GST in the digestive gland and gills of musselsMytilus galloprovincialis after 24 h (Canesi et al. 2010), while 0.001 mg mL-1 of CB NM decreased the AChE activity in gastrulae of sea urchin (Paracentrotus lividus) after 24 h (Mesaric et al. 2015b). In the present work, CAT and GST were not altered in the head of honeybees, which indicates that most probably no oxidative stress and detoxification process occurred. Also, the activity of AChE was not changed which is in contrast to the effect of ZnO NM previously reported for honeybees (Milivojevic et al. 2015). We can conclude that both TiO2 and CB NMs have low neurotoxic potential in honeybees. In the study of Milivojevic et al. (2015) the alteration of AChE was predominately explained as an effect of released Zn2+from ZnO NM. Nevertheless, in the study of Romih et al. (2015), the authors suggested that both dissolved ions and NMs in the subtoxic range could be responsible for the activation of different metabolic pathways in the hepatopancreases of crustacean Porcellio scaber. They have found that the ZnO NM induced different metabolic responses from those induced by Zn2+ salt. Based on this study it could be also the case with TiO2 and CB NMs but at the moment no data are available to support this theory. We can conclude that both TiO2 and CB NMs have low neurotoxic potential in honeybees. At the moment no data on the realistic environmental concentrations of nano TiO2 and nano CB are available. Predicted environmental concentration (PEC) for TiO2 NM was estimated at 107 mg kg-1 for wastewater treatment plant sludge, and 21 ng L-1 for surface waters (data from 2009) (Gottschalk 2009). TiO2 NM production market is constantly increasing and these NMs are expected to have higher release into soil, water and air in the future (Keller et al. 2013). The upper quantity of TiO2 NM estimated to pass through the waste water system in 2010 was nearly 48,000 t/year, with a potential for over 38,000 t/year to be added to the soil, and 32 000 t/year to landfill mainly through application of biosolids, but also small quantities through atmospheric deposition (1600 t/ year) (Keller et al. 2013). Carbon nanomaterials are also among those with high expected production, but their emission rates are lower compared to TiO2 52 Acta Biologica Slovenica, 59 (1), 2016 NM (e.g. 10 times lower for carbon nanotubes) (Keller et al. 2013). We were unable to find the PEC values for nano CB, but PEC values have been calculated for non-nano CB based on the data obtained in the period 1999-2010 (Screening Assessment for the Challenge 2013). The estimated PEC from industrial emission (inks and paints industry) was 6.6 mgL-1 for river, which is based on a total of4 336 447 kg of CB used/year. In conclusion, currently estimated PEC values for TiO2 and CB NMs are significantly lower than those tested in this study (1 mg mL-1). Future production and release rates of these two NMs are expected to be very high (Keller et al. 2013). Therefore it is reasonable to analyze the potential hazard of high TiO2 and CB NMs exposure concentrations (up to 1 mg mL-1). The current study reveals that both TiO2 and CB NM have no adverse effects, neither sub-lethal nor lethal, on honeybees after a 10-days exposure. In this regard, the use of carbon and titanium NMs in agriculture, instead of those with more hazardous potential, could have an advantage. Acknowledgements This investigation was supported by the Slovenian Research Agency, through Research program „Integrative zoology and speleobiology (P1-0184)". Povzetek Medonosne čebele (Apis mellifera) ogrožajo različni dejavniki, med katere lahko štejemo tudi povečano proizvodnjo in uporabo različnih nano-materialov v kmetijstvu v obliki nano-pesticidov in nano-gnojil. Vpliv kovin na čebele, sploh v obliki nanodelcev, je zelo slabo raziskan. Zato smo se odločili, da preučimo učinke dveh vrst nanomaterialov, ki veljata za dokaj inertna v smislu raztapljanja in interakcije z biološkimi sistemi: nano-črnega ogljika (nČO) in nano-titanovega dioksida (nTiO2). Odrasle čebele delavke kranjske čebele (Apis mellifera carnica) smo kronično (10 dni) hranili s suspenzijama nTiO2 (1 mg mL-1, n=40) ali nČO (1 mg mL-1, n=40), v 1.5 M raztopini saharoze. Kontrolna skupina čebel (n=40) je bila hranj ena samo z 1.5 M raztopino saharoze. Ugotovili smo, da 10-dnevno hranjenje z nTiO2 ali nČO ni vplivalo na preživetje ali stopnjo prehranjevanja čebel. Prav tako nTiO2 ali nČO nista vplivala na aktivnosti dveh antioksidativnih encimov, katalazo in glutation S-transferazo ter na aktivnost pokazatelja delovanja holinergičnega živčnega sistema encima acetilholinesteraza v možganih čebel. Ti rezultati kažejo na to, da uporabljeni nanomateriali verjetno niso imeli škodljivega učinka na čebele. Trenutno ocenjene predvidene vrednosti okoljskih koncentracij za nTiO2 in nČO so znatno nižje od teh, ki smo jih uporabili v sedanji raziskavi. Sklepamo, da je morebitna uporaba nTiO2 in nČO v kmetijstvu primernejša od tistih pripravkov, ki imajo več škodljivih učinkov na organizme. References Aruoja, V., Dubourguier, H., Kasemets, K., Kahru A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci Total Environ, 407, 1461-1468. Barata, C., Varo, I., Navarro, J.C., Arun, S., Porte C., 2005. 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Ambulanta za male, divje in eksotične živali, Glavni trg 7, 2366 Muta bVeterinarska fakulteta, Univerza v Ljubljani, Gerbičeva 60, 1000 Ljubljana c Trubarjeva 3, 3310 Žalec dFakulteta za naravoslovje in matematiko, Univerza v Mariboru, Koroška cesta 160, 2000 Maribor Correspondence: franc.janzekovic@um.si Izvleček: Na štirih skeletih sive čaplje (Ardea cinerea) smo proučili morfološke lastnosti kosti in skeletnih sklopov, predvsem nekatere segmente lobanje, hrbtenice, plečnega obroča, prsnice in medeničnega obroča. Za lobanjo je značilna izrazita kra-niofacialna upogibna cona (zonaflexoria craniofacialis), dolga čelnica (os frontale) z izrazito vzdolžno depresijo (depressio frontalis), dolg postorbitalni lok (arcuspostor-bitalis), obsežna senčnična jama (fossa temporalis) in dodatna plitka podsenčnična jama (fossa subtemporalis). Parasfenoidni ročaj (rostrumparasphenoidale) je izrazit, krilatki (ossa pterygoidea) pa močni in premi. Pri vratnih vretencih je značilna močnej ša kalcifikacija šestega vretenca in njegova toga povezanost s petim. V področju notarija (notarium) vretenca niso zraščena: med zadnjim vratnim in prvim prsnim vretencem je jasen sklep. Tudi prva tri prsna vretenca niso medsebojno zraščena, pač pa tesneje povezana v ventralnem delu. Četrto prsno vretence je izrazito pregibno (prosto), kot pri drugih ptičih. Peto prsno vretence je pridruženo sinsakrumu. Ključnici sta koščeno zraščeni v vilice furcula), njuna apofiza pa tvori sinostozo z vrhom prsničnega gredlja (apex carinae). Krokarnici se končujeta na prsnici vsaka v svojem sklepnem žlebu (sulcus articularis coracoideus), in sicer tako, da je sternalni konec desne krokarnice položen nekoliko preko leve. Na ročaju prsnice (manubrium sterni) manjka notranji trn (spina interna), na njegovem mestu je kratek žleb. Osnovni morfološki opis dopolnjujejo slike kosti oz. skeleta in dva rentgenska posnetka na živi živali, predstavljeni so tudi rezultati meritev posameznih kosti. Ključne besede: anatomija, skelet, ptiči, siva čaplja Abstract: In the skeletons of four grey herons (Ardea cinerea), we studied the morphological characteristics of bones and skeletal groups, particularly certain segments of the skull, spine, pectoral girdle, sternum and pelvic girdle. The skull is characterized by a pronounced craniofacial bending zone (zona flexoria craniofacia-lis), long frontal bone (os frontale) with a distinct longitudinal depression (depressio frontalis), long postorbital arch (arcus postorbitalis), extensive temporal fossa (fossa temporalis) and additional shallow subtemporal fossa (fossa subtemporalis). The parasphenoid rostrum (rostrum parasphenoidale) is distinctive, pterygoids (ossa 56 Acta Biologica Slovenica, 59 (1), 2016 pterygoidea) are strong and straight. Cervical vertebrae are characterized by stronger calcification of the sixth vertebra and its rigid link to the fifth. At the notarium region vertebrae are not fused: a joint is clearly seen between the last cervical and first thoracic vertebrae. The first three thoracic vertebrae are not fused, however, they are tightly connected at the ventral section. The fifth dorsal vertebra is associated with the synsacrum. The clavicles are fused into the fork (furcula) and their apophysis forms a synostosis with the top of the sternal keel (apex carinae). Coracoids end at the sternum, each in its own groove (sulcus articularis coracoideus), in a way that the sternal end of the right coracoid is placed slightly over the left. On the handle of the sternum (manubrium s. rostrum sterni) an internal thorn (spina interna) is missing, with a short groove in its place. The basic morphological description is complemented by images of bones or the skeleton, and two X-ray images of a live animal. Presented are also the measurements of individual bones. Keywords: anatomy, skeleton, birds, Ardea cinerea Uvod Siva čaplja (Ardea cinerea) je močvirski ptič iz družine čapelj (Ardeidae). Naseljuje Evropo, vzhodno Azijo, zahodno Kitajsko in Afriko. Odrasla žival tehta od 1000 do 2000 g (povprečno 1700 g), visoka je od 90 do 100 cm, razpon peruti meri od 175 do 190 cm. Dnevno potrebuje okoli 400 g hrane. Prehranjuje se z manjšimi vretenčarji, predvsem ribami, dvoživkami, mišmi in volu-haricami (Cramp, 1994). Družino Ardeidae smo še nedavno uvrščali v red močvirnikov (Ciconiiformes), ki je združeval v glavnem močvirske tropske ptiče z dolgimi nogami in dolgim kljunom, ki brodijo po vodi in plenijo stoje. Na podlagi analize DNK pa je mednarodni ornitološki kongres (IOC) prerazporedil družino čapelj v red veslonožcev (Pelecaniformes) (Gill in Donsker, 2010). Pri opisovanju in poimenovanju kosti in skeleta smo se naslonili predvsem na naslednjo literaturo: Furbringer (1888, 1902), Boas (1929, 1933), Payne in Risley (1976), King in McLel-land (1984), Rigler (1985, 1990), Kellner (1986), Baumel in sod. (1993), NAV (2012), Livezey in Zusi (2006) in Golob (2011). Pri izhodiščni raziskavi skeleta sive čaplje (Zajc, 2010) nismo našli ustrezne domače literature, v svetovni literaturi pa je več člankov, ki obravnavajo čaplje (Ardeidae). Posebej je treba omeniti primerjalno analizo čapelj, ki sta jo opravila Payne in Risley (1976) in morfometrični opis postkranialnega skeleta čapelj v doktorski disertaciji Kellner-jeve (1986). Posamezne lastnosti skeleta čapelj so opisane v Nomina Anatomica Avium (Baumel in sod., 1993) in v delu Livezey-a in Zusi-ja (2006). V okviru priprave članka o anatomskem poimenovanju kosti ptičev (Janžekovič in sod., 2015) smo opravili poglobljeno analizo skeleta sive čaplje. Pričakujemo, da bo vsebina članka koristila tudi v veterinarski praksi, saj so pri sivi čaplji razmeroma pogoste poškodbe z zlomi kosti. Namen raziskave je opisati anatomske značilnosti izbranih kosti sive čaplje, predvsem lobanje, hrbtenice in privesnega okostja, nekatere tudi izmeriti. Material in metode V raziskavo smo vključili štiri skelete sive čaplje, dva hrani Oddelek za biologijo, Fakulteta za naravoslovje in matematiko, Univerza v Mariboru, dva pa Prirodoslovni muzej Slovenije, Ljubljana. Posamezne kosti smo opisali, izmerili in fotografirali. Opis Opredelili in opisali smo posamezne kosti in dele skeleta. Pri tem smo se naslonili na literaturo, ki jo navajamo v uvodu. Golob et al.: Anatomske značilnosti skeleta sive čaplje 57 Merjenje Kosti smo merili s kljunastim merilom z natančnostjo 0,1 mm, daljše kosti (> 16 cm) pa z natančnostjo 1 mm. Morfometrične spremenljivke smo povzeli po Cohen-u in Serjeantson-u (1996), predstavljene so tudi v Zajc-evi (2010) diplomski nalogi. Izračunali smo povprečne vrednosti za posamezne kosti (x) in standardni odklon (SD). Fotografiranje Fotografije smo posneli s fotoaparatom Canon PowerShot SX120 IS v največji resoluciji 3648x2736 slikovnih pik in Canon EOS 350D digital. Pri fotografiranju smo uporabili temnejšo podlago, zaradi boljšega kontrasta. Fotografiranje je izvedel Z. Golob. Rentgen Rentgenske slike smo posneli z aparatom FUJIFILM FCR CAPSULA x Model CR-IR 357. Rezultati in razprava Rezultati so razvrščeni po sklopih: skelet glave, hrbtenica z rebri in prsnico, kosti plečnega obroča in peruti ter kosti medeničnega obroča in medeničnih okončin. Nekateri rezultati so opremljeni z izsledki v literaturi in kratko razpravo. Pri predstavitvi privesnega skeleta podajamo predvsem biometrične lastnosti. Skelet glave Dorzalna površina lobanje Lobanja (cranium) skupaj s kljunom (rostrum maxillae etmandibulae) meri v dolžino povprečno 19 cm, in sicer okrog 12,2 cm od konice kljuna do kraniofacialne upogibne cone (zona flexoria craniofacialis) in okrog 6,6 cm od upogibne cone do kavdalnega konca lobanje; povprečna višina lobanje je 3,6 cm (Tab. 1). Kljun je pri čaplji dolg in suličast (Sl. 1), krani-ofacialna upogibna cona pa se nahaja na začetku vzdolžne depresije čelnice (depressio frontalis). Zadnji del te depresije je v obliki medianega jarka, ki se končuje pri prečnem senčničnem grebenu (crista temporalis transversa; Baumel in sod., 1993), ki ga imenujemo tudi dorzalni senčnični greben (crista temporalis dorsalis; Livezey in Zusi, 2006). Od tod naprej teče sagitalni tilnični greben (crista nuchalis sagitalis), ki se konča pri prečnem tilničnem grebenu (crista nuchalis transversa). Od tega grebena proti veliki odprtini (foramen magnum) pa se nahaja sagitalna malomožganska štrlina (prominentia cerebellaris). Le-ta je pri sivi čaplji slabo izražena, opazen je le nizek greben te štrline. Dorzalni senčnični greben (crista temporalis dorsalis) je pri sivi čaplji zglajen (Sl. 1 in 3). Lateralno se končuje s postorbitalnim podaljškom (processus postorbitalis), ki je kratek. Od sagi-talnega tilničnega grebena in izza postorbitalnega podaljška se s temenice (os parietale) spušča proti senčnici (os squamosum s. temporale) žlebasta ugreznina, senčnična jama (fossa temporalis). Tabela 1: Meritve lobanje (cm) s srednjimi vrednostmi (x) in standardnim odklonom (SD). Table 1: Measurements of the skull (cm), with mean (x) and standard deviation (SD). Oznaka živali Specimen Dolžina kljuna Beak length Dolžina lobanje Skull length Celotna dolžina Total length Višina lobanje Skull height 1 14,1 6,8 20,9 3,2 2 11,2 6,2 17,4 3,3 3 10,5 6,0 17,0 3,9 4 13,1 7,2 20,5 3,8 r ± SD 12,2 ± 1,7 6,6 ± 0,6 19,0 ± 2,0 3,6 ± 0,4 58 Acta Biologica Slovenica, 59 (1), 2016 Slika 1: Lobanja sive čaplje z desne strani. Figure 1: The grey heron skull from right side. Legenda / Legend: cns = crista nuchalis sagitalis, cnt = crista nuchalis transversa, ctd = crista temporalis dorsalis, ctv = crista temporalis ventralis, d = dens, M = mandibula, Oq = os quadratum, pz = processus zygomaticus t = tomium, zf = zona flexoria craniofacialis. Kavdalno omejuje senčnično jamo spodnji senčnični greben (crista temporalis ventralis; Livezey in Zusi, 2006), ki se oddeli od prečnega tilničnega grebena (crista nuchalis transversa) in se končuje s kratkim ličničnim podaljškom (processus zygomaticus) (Sl. 3, pz). Ventrolateralno omejuje senčnično jamo rostralni greben senčnice (crista temporalis rostralis; Livezey in Zusi, 2006). Izrazi crista temporalis dorsalis, crista temporalis ventralis in crista temporalis rostralis so skladni s sedanjo anatomsko terminologijo in primernejši od izrazov crista temporalis superior, crista tem-poralis inferior in crista temporalis anterior, ki sta jih uporabila Pascotto-va in Donatelli (2003) pri opisu skeleta lobanje družine Momotidae. Izrazi anterior, posterior, superior in inferior se le izjemoma uporabljajo pri štirinožnih živalih (NAV, 2012). Pod senčnično jamo, na senčnici nad otičnim podaljškom kvadratne kosti, se nahaja plitka podsenčnična jama, fossa subtemporalis (Sl. 3, fst). Spodnja čeljustnica (mandibula) Mandibula sive čaplje ima dolg zobni (os dentale) oz. simfizni del (pars symphysialis), ki predstavlja osnovo obeh mandibularnih vej (rami mandibulae); sledita vmesni (pars intermedia) in kavdalni del (pars caudalis). Obe čeljustnici sta ravni, v sprednjem, tj. zobnem področju pa je mandibula nekoliko usločena, manj izrazito tudi maksila (Sl. 1). Na tem mestu je opaziti med čeljustnicama manjšo zev. Rožena prevleka (ram-photeca) tvori na mandibuli in maksili zobčast 1 do 2 mm visok roženi tomij (tomium) (Sl. 1, t). Zobci tomija so v področju usločenih delov čeljustnic izraziti in kavdalno usmerjeni. Na konici mandibule tvori ramfoteka oster koničast zaključek, medtem ko je pri maksili le-ta zadebeljen v »zob« z ostrim koncem (Sl. 1, d). Močno keratinizirana ramfoteka se razteza od vrha rostrumov v kavdalni smeri do ravni mandibularnega kota (angulus mandibularis). Na svojem kavdalnem koncu, nasproti kvadratne kosti, se veji mandibule razširita in tvorita dva medsebojno povezana podaljška, medialnega (processus medialis) in lateralnega Golob et al.: Anatomske značilnosti skeleta sive čaplje 59 (processus lateralis) (Sl. 2, pmm, pml). Med njima je kotanja (fossa articularis quadratica), ki služi artikulaciji s čvrši mandibulamega podaljška (processus mandibularis) kvadratne kosti (os quad-ratum) (Sl. 1 in 3). Sklepne ploskve oz. ponvice (cotylae fossae articularis) so pri sivi čaplji tri: medialna, lateralna in kavdalna (Sl. 2, cm, cl, cc). Pod medialno ponvico (cotyla medialis) se od medialne strani v notranjost sklepne kotanje razteza žleb (sulcus intercotylaris), pred njim pa je poševen greben (crista intercotylaris) z grbico (tuberculum intercotylare). Za medialno ponvico se nahaja sklepna zračna odprtina (foramen pneumaticum articulare, Sl. 2, fpa). Končni del mandibule, za sklepnim področjem s kvadratno kostjo, je značilno podkvasto podaljšan (Sl. 2 in 3), dorzokavdalna površina tega podaljška pa je nekoliko ugreznjena (fossa caudalis s. posterior, Sl. 3, fc). Ta jama služi pripetju mišice spuščevalke mandibule (musculus depressor mandibulae), ki ima svoje izhodišče v podsenčični jami (fossa subtemporalis) med prečnim tilničnim grebenom in senčnično jamo (Baumel in sod., 1993). Na meji s sklepnim delom mandibule se nahaja prečen greben (crista transversa fossae), ki se razteza med medialnim in lateralnim podaljškom mandibule (Sl. 2). Na grebenu se nahaja grbica (tuberculum). Rostralno od sklepnega področja je na medialni strani mandibularne veje ovalnajama (fossa medialis mandibulare) (Sl. 2, fmn), ki ima na sprednjem koncu odprtino v mandibularni kanal; imenujemo jo tudi vhodna kotanja nevrovaskularnega kanala (fossa aditus canalis neurovascularis) (Baumel in sod., 1993). Na kavdalnem koncu ventralnega roba te kotanje se nahaja psevdotemporalna grbica (tuberculum pseudotemporale), nasadiščno mesto za tetivo psevdotemporalne površinske mišice (m. pseudotemporalis superficialis) (Baumel in sod., 1993). Slika 2: Mandíbula sive caplje z dorzalne strani, v okviru kavdalni del z laterodorzalne strani. Figure 2: The grey heron mandible, dorsal view. In the circle is enlarged caudal part of the mandible, laterodorsal view. Legenda / Legend: cc = cotyla caudalis, cint = crista intercotylaris, cl = cotyla lateralis, cm = cotyla medialis, ctf = crista transversa fossae, faq = fossa articularis quadratica, fmm = fossa medialis mandibulae, fpa = foramen pneumaticum articulare, pcm = pars caudalis mandibulae, pml = processus mandibulae lateralis, pmm = processus mandibulae medialis, procc = processus coronoideus, psm = pars symphysialis mandibulae. 60 Acta Biologica Slovenica, 59 (1), 2016 Kvadratna kost (os quadratum) Kvadratna kost je osrednja sestavina skeleta lobanje v lateroventralnem področju (Sl. 1 in 3). Sestoji iz telesa in treh glavnih podaljškov, ti so mandibularni (processus mandibularis), orbitalni (processus orbitalis) in otični podaljšek (processus oticus). Na mandibularnem podaljšku so štirje sklepni čvrši, in to trije na ventralni površini za stik z mandibulo (condylus caudalis, condylus lateralis in condylus rostralis) in eden kraniome-dialno za stik s krilatko (condylus pterygoideus). Kraniolateralno se na tem podaljšku nahaja sklepna ponvica (cotyla quadratojugalis), za sklep s kvadratojarmno kostjo (Sl. 3, glava puščice). Otični podaljšek ima dva sklepna čvrša, za stik s senčnico in z otičnim stebričkom (pila otica; op.: kompleks zlitih otičnih koščic, ki vsebuje med drugim koščeni labirint notranjega ušesa; Baumel in sod., 1993). Končni del orbitalnega podaljška se lopatasto razširi in se prislanja na orbito, na katero se rahlo pripenja. Na posušenem skeletu je orbitalni podaljšek dejansko nekoliko odmaknjen od podlage. Slika 3: Zadnji del lobanje in spodnje čeljustnice z desne strani. Glava puščice označuje sklep med kvadratno kostjo in kvadratnojarmno kostjo. Figure 3: Posterior part of the skull and mandible from the right side. The arrow-head indicates the joint between quadrate and quadratojugal bones. Legenda / Legend: ano = apertura nasi ossea, cnt = crista nuchalis transversa, ctd = crista temporalis dorsalis, ctv = crista temporalis ventralis, fc = fossa caudalis s. posterior, fena = fenestra antorbitalis, fonti = fonticulus interorbitalis, fonts = fonticulus supraorbitalis, ft = fossa temporalis, fst = fossa subtemporalis, Ol = os lacrimale, On = os nasale, Omes = os mesethmoidale, Oqj = os quadratojugale, pectl = processus ectethmoidalis lateralis, pol = processus orbitalis lacrimalis, ppo = processus postorbitalis, pz = processus zygomaticus. A, B, C = os quadratum : A = processus oticus, B = processus orbitalis, C = processus mandibularis. Golob et al.: Anatomske značilnosti skeleta sive čaplje 61 Krilatka (os pterygoideum) in parasfenoidni ročaj (rostrum parasphenoidale) Krilatka je v paru, z značilnim položajem na spodnji površini lobanje (Sl. 4, Optd, Opts). Na koncu proti mandibularnemu podaljšku kvadratne kosti ima sklepno ponvico, pri nebnici (os palatinum) pa tvorita obe veji obsežnejše sklepno področje skupaj s parasfenoidnim ročajem (rostrum parasphenoidale) (Sl. 4, puščica). Pri čaplji je krilatka iztegnjena (ravna), medtem ko je pri nekaterih ptičih lokasta (npr. Mergus) ali upognjena (npr. Vanellus) (Baumel in sod., 1993). Za parasfenoidnim ročajem se nahaja parasfenoidna plošča (lamina parasphenoidalis), ki sega do podnožja zatilničnega čvrša (condylus occipitalis) (Sl. 4, co). To podnožje je pravzaprav ozek pas bazilame zatilnice (os basioccipitale). Končni del parasfenoidne plošče pokriva mamilarna hribčka (tuberculi basilare), ki sta na rostralnih kotih bazilarne zatilnice. Slika 4: Lobanja z ventralne strani. Puščica označuje sklepno področje med krilatkama in nebnicama. Figure 4: The gray heron skull from ventral side. Arrow indicates the joint between palatines and pterygoids. Legenda / Legend: aj = arcus jugale, co = condylus occipitalis, fm = foramen magnum, lp = lamina parasphenoidalis, Opd = ospalatinum dextrum, Ops = ospalatinum sinistrum, Optd = ospterygoideum dextrum, Opts = os pterygoideum sinistrum, Oq:porb = os quadratum: processus orbitalis, rp = rostrum parasphenoidale. Hrbtenica (columna vertebralis) Hrbtenica čaplje sestoji - kot pri drugih ptičih - iz vratnih in prsnih vretenc, sinsakruma, prostih repnih vretenc in pigostila. Najznačilnejše je področje vratu. Vrat je dolg in pri odraslih živalih esasto ukrivljen tudi med letom, pri lovljenju plena pa ga žival lahko sunkovito iztegne. Vratna vretenca (vertebrae cervicales) Vse proučene živali so imele po 19 vratnih vretenc. Predzadnje in zadnje nosita par nepopolnih reber (costae incompletae), s to razliko, da imata rebri zadnjega vratnega vretenca kavljasti podaljšek (processus uncinatus), rebri predzadnjega pa ne (Sl. 5). Skladno z opredelitvijo v NAA (Baumel in sod., 1993) smo za prvo prsno vretence smatrali tisto, ki nosi par popolnih pravih reber (costae completae verae) (Sl. 5, dvoglava puščica). 62 Acta Biologica Slovenica, 59 (1), 2016 Boas (1929) je pri sivi čaplji ugotovil 16 oz. 17 vratnih vretenc. Razliko med njegovo in našo navedbo je mogoče pripisati drugačnemu metodološkemu kriteriju; domnevamo, da je Boas zadnji dve vratni vretenci štel med prsna. Boas je vratni del hrbtenice razdelil v tri segmente in v drugem segmentu ugotovil skupino sublateralno zraščenih vretenc. Pri preverjanju njegove trditve smo na rentgenskih posnetkih vratu ugotovili močnejšo kalcifikacijo pri šestem vratnem vretencu (Sl. 6) in pri skupini vretenc od 12-tega do 16-tega vretenca (Sl. 7). Potrebne so nadaljnje raziskave vratnega področja hrbtenice pri sivih čapljah različne starosti. Slika 5: Zadnja tri vratna vretenca, prsna vretenca, medenični obroč, prosta repna vretenca, pigostil, plečnica, krokarnici, furkula, rebra in prsnica, slikano z leve strani. Enoglava puščica označuje glavico nepravega rebra, dvoglava puščica prvi par pravih reber, glava puščice pa sklep med furkulo in vrhom gredlja. Figure 5: The last three cervical vertebrae, thoracic vertebrae, pelvic girdle, free caudal vertebrae, pygostyle, scapula, coracoid, furcula, ribs and keel from the left view. One-headed arrow marks the head of the false rib, two-headed arrow the first pair of true ribs, and the arrow-head the joint between furcula and the tip of the keel. Legenda / Legend: aip = articulatio ischio-pubica, Cd = coracoideum dextrum (sternalni konec desne krokarnice, ki deloma sega preko leve), Ci = costa incompleta, Cs= coracoideum sinistrum, Csp = costa spuria, cst = carina sterni, F = furcula, l = incisura sterni, P = pygostylus, pi = processus interclavicularis, sex = spina externa, VC17, VC18, VC19 = vertebra cervicalis 17, 18, 19, VT1, VT2, VT3, VT4, VT5 = vertebra thoracalis 1, 2, 3, 4, 5, * = processus craniolateralis sterni. Golob et al.: Anatomske značilnosti skeleta sive čaplje 63 Slika 6: Rentgenski posnetek lobanje in prvih šest vratnih vretenc z ventralne strani. Odrasla žival. Opazna je tesna povezanost med petim in šestim vretencem. Figure 6: Radiograph of the skull and first six cervical vertebrae from the ventral side. An adult specimen. A close relationship between the fifth and sixth vertebrae is visible. Legenda / Legend: A = atlas, VC6 = vertebra cervicalis 6, zf = zonaflexoria craniofacialis. Slika 7: Rentgenski posnetek trupa z dorzalne strani. Izrazitejša kalcifikacija se nahaja na področju od 12. do 16. vratnega vretenca (glava puščice) in prsnih vretenc. Notarija ni mogoče jasno opredeliti: medvretenčni sklepi se lepo vidijo (puščica: sklep med zadnjim vratnim in prvim prsnim vretencem); 1, 2, 3, 4, 5 = prvo, drugo, tretje, četrto in peto prsno vretence. Figure 7: Radiograph of the torso from the dorsal side. Pronounced calcification is located in the area from 12th to 16th cervical vertebrae (the arrow-head) and thoracic vertebrae. The notarium cannot be clearly defined: intervertebral joints are visible (arrow: joint between the last cervical and first thoracic vertebra); 1, 2, 3, 4, 5 = the first, second, third, fourth and fifth thoracic vertebrae. 64 Acta Biologica Slovenica, 59 (1), 2016 Prsna vretenca (vertebrae thoracicae) Prsni del hrbtenice sestoji iz petih vretenc. Zadnje vratno in prva tri prsna vretenca so pri mnogih ptičih vrstah zlita in tvorijo enoto notarij (notarium). Pri sivi čaplji je stanje nekoliko drugačno, kajti med zadnjim vratnim in prvim prsnim vretencem se nahaja izrazit sklep (Sl. 7; puščica). Lateroventralni deli prvih treh prsnih vretenčnih teles so tesneje medsebojno povezani, trnaste podaljške (processus spinosi) pa pokriva močna vezivna opna, ki sega od zadnjega vratnega vretenca do preacetabularnega krila črevnic. Notarijevo področje čaplje se torej razlikuje od tipičnega notarija, saj manjkajo koščene medvretenčne povezave med vrhovi trnastih podaljškov, med stranskimi podaljški in na ven-tralni strani. Na rentgenskih posnetkih je sicer opaziti pri vretencih notarijevega področja nekoliko izrazitejšo kalcifikacijo, a tudi medvretenčne sklepe (Sl. 7). Menimo, da je med vretenci notarija sive čaplje pričakovati določeno pregibnost. Četrto prsno vretence je prosto. Prsna vretenca tvorijo sklepe s popolnimi rebri (costae complete) in sicer prva štiri s pravimi rebri (costae completae verae), peto, ki spada v sinsakrum, pa ima par popolnih nepravih reber (costae completae spuriae). Slednji nimata kavljastega podaljška, njun vertebralni del pa je prek rebrne grčice (tuberculum) zraščen s prečnim podaljškom (processus transversus) petega prsnega vretenca in ta s preacetabularnim delom črevnice (Sl. 9). Sinsakrum (synsacrum) Preacetabularni del sinsakruma sestoji iz enega prsnega in štirih ledvenih vretenc, dve ali tri križna vretenca oblikujejo acetabularni del sinsakruma in šest zlitih repnih vretenc postacetabularni del sinsakruma. Boas (1933) navaja pri sinsakrumu sive čaplje skupno 14 ali 15 zraščenih vretenc. Med zadnjim prsnim in prvim ledvenim vretencem je pri mlajših živalih opaziti sklepno progo, medtem ko so naslednja vretenca sinsakruma že ankilo-zno zraščena. Sinsakrum je sinostozno zraščen s kolčnicama (ossa coxae). Prosta repna vretenca (vertebrae caudales liberae) Prostih repnih vretenc je pet, ki imajo dobro razvite prečne podaljške (Sl. 9 in 10). Pigostil (pygostylus) Pigostil ali repni opornik je enojna in skoraj pravokotno navzgor obrnjena kost (Sl. 5). Oblikuje bazo (basis pygostyli), ki ima na kranialnem koncu sklepno ploskev facies articularis cranialis) za sklep z zadnjim prostim repnim vretencem. Plošča pigostila (lamina pygostyli) meri pri sivi čaplji 15 x 7 mm. Rebra (costae) Zadnji dve vratni vretenci nosita para nepopolnih reber (costae incompletae) (slika 5); pri rebrih zadnjega vratnega vretenca je prisoten kavljasti podaljšek (processus uncinatus). Prsna vretenca nosijo popolna prava rebra (costae completae verae) in prvo ledveno popolni nepravi rebri (cos-tae completae spuriae), ki pa nimata kavljastega podaljška (Sl. 5, Csp). Prsnica (sternum) Prsnicaje dobro razvita in z izrazitim gredljem (carina sterni) (Sl. 5, cst). Pri odrasli čaplji meri v dolžino 9 cm. Njen kranialni konec predstavlja rebrni del (costosternum) in kavdalni konec ksifoidni del (xiphosternum ali metasternum) (Furbringer, 1888). Na kranialnem koncu prsnice se nahajata sklepna žlebova za krokarnici (sulci articula-ris coracoidei) (Sl. 8, sacd/sacs). Pri čaplji se krokarnici v svojem distalnem delu ne končujeta v skupni ravnini, ampak se nekoliko prekrižata: desna krokarnica je pomaknjena deloma preko leve in njen konec je zato nekoliko ventralnejši od konca leve krokarnice. Takšno oz. podobno umestitev krokarnic na prsnici so opisali tudi pri turakih (Musophagidae) (Furbringer, 1888), pri nekaterih cevonoscih (Procellariiformes) ter pri Buteo, Falkoniformes, Bubo in Ichtyornis (Baumel in sod., 1993). Golob et al.: Anatomske značilnosti skeleta sive čaplje 65 Slika 8: Prsnica s sprednje strani. Posebej so označeni sklepni ploskvi na notranji ustni (*), žleb v mediani ravnini (glava puščice), krokarnična sklepna žleba (sacd, sacs), zunanji trn (sex) kljuna ali ročaja prsnice (rostrum s. manubrium sterni) in (hrapava) sklepna ploskev za vilice (faf). Figure 8: Sternum - frontal view. Legenda / Legend: faf = facies articularisfurculae, pcl = processus craniolateralis sterni, sacd / sacs = sulcus articularis coracoideus dexter / sinister, sex = spina externa rostri. Pod sklepnim področjem s krokarnicama se v kranialni smeri razteza okrog 8 mm dolg bilateralno stisnjen podaljšek, zunanji trn (spina externa) (Sl. 5 in 8, sex), ki predstavlja najizrazitejši del prsničnega kljuna ali ročaja (rostrum s. manubrium sterni). Notranji trn (spina interna) pri čaplji manjka, na njegovem mestu pa se nahaja zareza oz. kratek žleb (Sl. 8). Grebenasta zunanja ustna (labrum externum) se nahaja pred umestitvijo končnega dela krokarnic v prečna sklepna žlebova (sulci articularis coracoidei) in se razteza lateralno od zunanjega trna. Nad sklepnim žlebom za krokar-nico je notranja ustna (labrum internum). Levi in desni del notranje ustne oblikujeta posebno ovalno in na medialnem koncu nekoliko razpotegnjeno sklepno ploskev za krokarnico (Sl. 8, zvezdici). Dorzalna ali visceralna površina prsnice je vzdolžno kotanjasta, obsega pa srčni in jetrni del (pars cardiaca et pars hepatica). Pred srčnim delom se nahaja obsežnejša zračna odprtina (foramenpneumaticum). Za sklepnim področjem s krokarnicama se strani prsnice dvigata v izrazit kraniolateralni podaljšek (processus craniolateralis) in se nato spustita v sklepno področje z rebri. V tem sklepnem področju so pri čaplji po štiri sklepne ponvice za sklep s sternalnim koncem popolnih pravih reber. Pri opazovanju s strani vidimo, da se omenjene sklepne ponvice nahajajo na kratkih prsnorebrnih sklepnih podaljških (proc-cessus articularis sternocostalis), pred vsakim sklepnim podaljškom pa je majhna a izrazita zareza z globeljo. Rebrni rob prsnice (margo costalis sterni) se od sklepnega področja z rebri nadaljuje v ksifoidni del prsnice, ki je na kavdalnem koncu zarezan v obliki črke V (Sl. 5). Gledano od zadaj (dorzokavdalno oz. ventralno) vidimo obe zarezi, levo in desno, ki spominjata na črko W. Apendikularni skelet (skeleton appendiculare) Plečni obroč (ossa cinguli) Plečnica (scapula) je izrazito sabljaste oblike (Sl. 5) in sega kavdalno do sprednjega roba črevnice. Povprečna dolžina obeh plečnic je 8,4 cm 66 Acta Biologica Slovenica, 59 (1), 2016 (Tab. 2 in 15). Skupaj s krokarnico in dorzalnim okrajkom ključnice (extremitas omalis claviculae) sooblikuje trikostni kanal. Tabela 2: Meritve na plečnici (scapula; v cm); izračun povprečnih vrednosti (x) in standardnega odklona (SD) za levo in desno plečnico ter za obe skupaj. Table 2: Measurements of the scapula (cm), with mean (x) and standard deviation (SD). L-left, D-right scapula. Živali Specimen Celotna dolžina Total length Največja kranialna diagonala Maximal cranial diagonal 1 8,8 8,8 1,6 1,6 2 8,5 8,6 1,4 1,4 3 7,7 7,9 1,4 1,5 4 8,6 8,5 1,6 1,5 X 8,4 8,5 1,5 1,5 SD 0,5 0,4 0,1 0,1 L + D 8,4 ± 0,4 1,5 ± 0,1 L D L D Ključnici (claviculae) sta ventralno v apofiznem področju koščeno zraščeni v vilice ali furkulo (furcula), končni del vilic pa oblikuje sklep s konico gredlja (apex carinae) (Sl. 5). Furbringer (1902) je opisal ta sklep kot sinostozo. Pri sivi čaplji opazimo v apofiznem delu vilic (apophysis furculae) izrazit medključnični podalj šek (processus interclavicularis), ki meri okrog 7 x 2 mm in se razteza proksimalno v kot med obema vejama furkule (Sl. 5, pi). Dorzalni konec ključnice ima podaljšek (processus acrocoracoideus) in sklepno ploskev za stik s krokarnico (facies articularis acrocoracoidea). Ostrčev podaljšek ključnice (processus acromialis claviculae) oblikuje sklepni stik z ostrcem plečnice (acromion). Krokarnica (os coracoideum) je krepka kost, dolga okrog 6,9 cm (Tab. 3 in 15). Dorzalni konec (extremitas omalis coracoidei) je s sklepi in vezmi čvrsto povezan s plečnico, vilicami in nadlahtnico, ventralni konec (extremitas sternalis coracoidei) pa s prsnico. Krokarnici sta v sklepnem področju s prsnico poravnani tako, da sega desna nekoliko navzpred od leve (Sl. 5, Cd), vsaka paje umeščena v svoj sklepni žleb na prsnici (sulcus articularis coracoideus) (Sl. 8, sacd/sacs). Tabela 3: Meritve leve in desne krokarnice (os coracoideum): njene celotne dolžine, bazalne širine, širine sklepne površine in dolžine njene medialne strani (v cm), posebej za levo in desno ter za obe skupaj (L+D). Table 3: Measurements of the coracoid (cm), with mean (x) and standard deviation (SD). L-left, D-right coracoid. Živali Specimen Celotna dolžina krokarnice Total lenght Bazalna širina Basal breadth Širina sklepne površine Breadth of the joint surface Dolžina med. strani Length of the medial side L D L D L D L D 1 7,1 6,8 3,0 3,0 2,3 2,2 6,5 6,4 2 6,7 6,7 2,6 2,8 2,0 2,1 6,1 6,0 3 6,8 6,8 2,3 2,4 2,0 2,1 5,8 5,9 4 7,1 7,2 2,1 2,2 2,7 2,7 6,3 6,2 X 6,9 6,9 2,5 2,5 2,3 2,3 6,2 6,1 SD 0,2 0,2 0,4 0,4 0,3 0,3 0,3 0,2 L + D 6,9 ± 0,2 2,6 ± 0,4 2,3 ± 0,3 6,2 ± 0,2 Golob et al.: Anatomske značilnosti skeleta sive čaplje 67 Kosti prsnih okončine (ossa alae) Pri čaplji je nadlahtnica (humerus) dolga, saj meri pri odrasli živali okrog 17,6 cm (Tab. 4). Na proksimalnem koncu nadlahtnice se nahaja glava (caput humeri) in dobro razvit prsni greben (crista deltopectoralis) za nasadišče prsnih mišic (kranialna površina). Na kavdalni površini glave nadlahtnice leži odprtina (foramen pneumaticum), skozi katero vstopa stranski betič ključnične zračne vrečke. Koželjnica (radius) je vzdolžno usločena in meri okrog 20 cm (Tab. 5). Komolčnica (ulna) je prav tako usločena in meri okrog 21 cm (Tab. 6). Njen ventralni rob ima papile za nasadišče vezi peresnih čebulic sekundarnih letalnih peres (papillae remigales ventrales), ki jih je pri sivi čaplji 12. Kavdalni konec koželjnice in komolčnice se v sklepu povezuje z nadlahtnico, kranialni pa oblikuje sklepno povezavo z zapestjem. Zapestnici (ossa carpi), koželjnična zapestnica (os carpi radiale) in komolčična zapestnica (os carpi ulnare) sta enako dolgi, in to 1,1 cm. Zapestnodlančna kost (carpometacarpus) je dolga 9,3 cm (Tab. 7). Dolžina prstnic (ossa digitorum manus) je predstavljena v preglednici (Tab. 8). Kostna osnova iztegnjene peruti meri pri sivi čaplji okrog 54 cm. Tabela 4: Meritve nadlahtnice (humerus): njene dolžine, največje širine na proksimalnem koncu, najmanjše širine telesa in največje širine na distalnem koncu (v cm), posebej za levo in desno ter za obe skupaj (L+D), z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 4: Measurements of the humerus (cm), with mean (x) and standard deviation (SD). L-left, D-right humerus. Živali Specimen Celotna dolžina nadlahtnice Total lenght Največja širina proksimalnega konca Maximal breadth of the proximal end Najmanjša širina telesa Minimal breadth of the corpus Največja širina dist. konca Maximal breadth of the distal end L D L D L D L D 1 17,7 18,0 2,7 2,7 1,0 1,0 2,4 2,5 2 17,5 17,5 2,6 2,6 1,0 1,0 2,2 2,2 3 16,7 16,8 2,4 2,5 0,9 0,9 2,1 2,1 4 18,1 18,4 2,6 2,5 0,9 1,0 2,3 2,1 x 17,5 17,7 2,6 2,6 1,0 1,0 2,3 2,2 SD 0,6 0,7 0,1 0,1 0,1 0,1 0,1 0,2 L + D 17,6 ± 0,6 2,6 ± 0,1 1,0 ± 0,1 2,2 ± 0,2 Tabela 5: Meritve koželjnice (radius): njene dolžine, najmanjše širine debla in največje širine na distalnem koncu (v cm), posebej za levo in desno ter za obe skupaj (L + D), z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 5: Measurements of the radius (cm), with mean (x) and standard deviation (SD). L-left, D-right radius. Žival Celotna dolžina Total lenght Najmanjša širina debla Minimal breadth of corpus Največja distalna širina Maximal distal breadth specimen L D L D L D 1 20,1 20,1 0,4 0,4 1,3 1,2 2 19,7 19,6 0,4 0,4 1,1 1,1 3 19,5 19.4 0,4 0,4 1,0 1,0 4 20,3 20,2 0,4 0,5 1,0 1,1 x 19,9 19,8 0,4 0,4 1,1 1,1 SD 0,4 0,4 0,0 0,1 0,1 0,1 L+D 19,9 ± 0,4 0,41 ± 0,04 1,1 ± 0,1 68 Acta Biologica Slovenica, 59 (1), 2016 Tabela 6: Meritve komolčnice (ulna): njene dolžine, največje širine na proksimalnem koncu, diagonalne širine proksimalnega konca, najmanjše širine telesa ter diagonalne širine distalnega konca (v cm), posebej za levo in desno ter za obe skupaj (L + D), z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 6: Measurements of the ulna (cm), with mean (x) and standard deviation (SD). L-left, D-right ulna. Žival Specimen Dolžina Total lenght Širina proksimalnega konca Breadth of the proximal end Diagonala proks. konca Diagonal of the proximal end Najmanjša širina telesa Minimal breadth of the corpus Diagonala distal. konca Diagonal of the distal end L D L D L D L D L D 1 20,8 21,0 1,7 1,7 1,9 1,8 0,7 0,7 1,3 1,3 2 20,6 20,5 1,5 1,5 1,8 1,7 0,6 0,6 1,3 1,3 3 20,3 20,2 1,5 1,6 1,2 1,3 0,5 0,7 1,3 1,3 4 21,4 21,5 1,6 1,6 1,5 1,5 0,6 0,7 1,3 1,4 x 20,8 20,8 1,6 1,6 1,6 1,6 0,6 0,7 1,3 1,3 SD 0,5 0,6 0,1 0,1 0,3 0,2 0,1 0,1 0,0 0,1 L+D 20,8 ! ± 0,5 1,6 ± 0,1 1,6 ± 0,3 0,6 ± 0,1 1,3 ± 0,04 Tabela 7: Meritve zapestnodlančnice (carpometacarpus): njene dolžine, dolžine manjše dlančnice, največje širine proksimalnega konca in diagonalne širine na distalnem koncu (v cm); posebej za levo in desno ter za obe skupaj (L + D), z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 7: Measurements of the carpometacarpus (cm), with mean (x) and standard deviation (SD). L-left, D-right carpometacarpus. Žival Specimen Skupna dolžina Total length Dolžina manjše dlančnice Length of the minor metacarpal Največja proks. širina Maximal proximal breadth Diagonala distalnega konca Diagonal of the distal end L D L D L D L D 1 9,6 9,6 9,5 9,5 1,7 1,8 1,4 1,3 2 9,2 9,2 9,1 9,1 1,6 1,6 1,2 1,2 3 8,9 8,9 8,7 8,7 1,3 1,4 1,1 1,0 4 9,7 9,6 9,3 9,2 1,6 1,7 1,2 1,1 x 9,4 9,3 9,2 9,1 1,6 1,6 1,2 1,2 SD 0,4 0,3 0,3 0,3 0,2 0,2 0,1 0,1 L+D 9,3 ± 0,3 9,1 ± 0,3 1,6 ± 0,2 1,2 ± 0,1 Tabela 8: Dolžina bazalne in terminalne prstnice večjega in dolžina prstnice manjšega prsta peruti (v cm); posebej za levo in desno stran ter za obe skupaj (L + D), z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 8: Measurements of the basal and terminal phalanx of the major digit and length of the minor digit (cm), with mean (x) and standard deviation (SD). L-left, D-right digit. Žival Specimen Večji prst: bazalna prstnica Basal phalanx of the major digit Večji prst: terminalna prstnica Terminal phalanx of the major digit Dolžina manjšega prsta Length of the minor digit L D L D L D 1 3,5 3,4 3,1 3,1 1,6 1,6 2 3,1 3,1 2,7 2,7 1,4 1,4 3 3,0 3,1 2,6 2,7 1,1 1,0 4 3,4 3,3 2,9 3,0 1,3 1,3 x 3,3 3,2 2,8 2,9 1,4 1,3 SD 0,2 0,2 0,2 0,2 0,2 0,3 L+D 3,2 ± 0,2 2,9 ± 0,2 1,3 ± 0,2 Golob et al.: Anatomske značilnosti skeleta sive čaplje 69 Kosti medeničnega obroča in okončin fossa cinguli membri pelvici) Medenica (pelvis) Pri odrasli sivi čaplji so medenične kosti črevnica (ilium), sednica (ischium) in dimeljnica (pubis) zlite v kolčnico (os coxae). Kolčnici sta zrasli s sinsakrumom pa tudi druga z drugo v področju preacetabularnih kril, nad dorzalnim grebenom sinsakruma (Sl. 9). Zraslost s sinsakru-mom je temeljita, tako da črevničnokrižničnega šiva (sutura iliosynsacralis) v področju postac-etabularnih kril črevnic ni opaziti. Ta šiv je npr. pri kokoši in puranu izrazit. Se pa na mestu zrasti s kavdalnim koncem sinsakruma, med prečnimi podaljški zlitih vretenc sinsakruma in krilom črevnice (tj. na mestu omenjenega šiva), nahaja sinostozen sklep (synostosis iliosynsacralis), ki oblikuje greben (Sl. 9, sis). V tem področju so pri sinsakrumu tri odprtine, foramina intertrans-versaria. Sicer pa se omenjeni greben nadaljuje s presledki prek medvretenčnih prostorov na prečnih podaljških prvih štirih prostih repnih vretenc (Sl. 9, dvoglava puščica). Pri čaplji je črevnična dorzalna kotanja preacetabularnih kril (fossa iliaca dorsalis) plitka, plitka je tudi kavdalna črevnična kotanja (fossa iliocaudalis) na koncu postacetabularnih kril črevnice. Črevničnosednična odprtina (foramen ilioischiadicum) je pri sivi čaplji ovalna, zadelana odprtina (foramen obturatum) pa se v kavdalni smeri nadaljuje v podaljšano sedničnodimeljnično okno fenestra ischiopubica) (Sl. 5). Medenica sive čaplje je razmeroma ozka, v področju sinsakruma meri v dolžino okrog 8,0 cm in v širino okrog 4,0 cm, zato sta tudi kotanji za umestitev ledvic ozki in razpotegnjeni. Kotanja na ventralni strani preacetabularnih kril (fossa iliaca ventralis) (Sl. 10, fiv) omogoča umestitev krani-alnega dela ledvic. V področju postacetabularnih kril črevnice je ledvična kotanja (fossa renalis) (Sl. 10, fr), ki je iz dveh z žlebom povezanih delov, manjšega kranialnega in obsežnejšega kavdalnega. Kranialni del (pars ischiadica fossae) se nahaja v področju acetabuluma in kavdalni del (pars pudenda fossae) v področju črevničnosednične odprtine (foramen ilioischiadicum). Slika 9: Področje sinsakruma, kolčnic, prostih repnih vretenc in pigostila z dorzalne strani. Sklepna odprtina kolčnice (fa) predeli medenični obroč na preacetabularni, acetabularni in postacetabularni del. Preacetabularni krili črevnice sta medsebojno zrasli, postacetabularni krili pa tvorita s sinsakrumom črevničnosinsakralno sinostozo (sis). Puščici: prečni podaljšek vretenc (processus transversus), glava puščice: fossa iliaca dorsalis. Figure 9: The synsacrum area, hip, free caudal vertebrae and pygostyl; dorsal view. Legenda / Legend: apoi = ala postacetabularis ilii, apri = ala preacetabularis ilii, fa = foramen acetabuli, sis = synostosis iliosynsacralis, VC1, VC2, VC3, VC4, VC5 = vertebra caudalis libera 1, 2, 3, 4, 5. Arrows = processus transversus, Head of arrow = fossa iliaca dorsalis. 70 Acta Biologica Slovenica, 59 (1), 2016 tt Slika 10: Obroč medenične okončine z ventralne strani, v okviru področje tretjega, četrtega in petega prsnega vretenca. Izrazitejši globeli sta ventralna črevnična jama (fiv) in ledvična jama (fr), slednjaje predeljena na kranialni sednični del (pars ischiadica fossae) in kavdalni dimeljni del (pars pudenda fossae). Prosta repna vretenca (VC1, 2, 3, 4, 5). V okviru: Nepravo popolno rebro z glavico (cc) in grčico (tc). 3, 4, 5 = tretje, četrto (prosto) in peto prsno vretence, temna glava puščice = sklep glavice rebra (cc) s telesom vretenca (corpus vertebrae) na lateralni površini vretenca, svetla glava puščice = sklep kvrgice rebra (tc) s prečnim podaljškom (proct) vretenca. Figure 10: The pelvic girdle from ventral side with the third, fourth and fifth thoracic vertebrae in the circle. Legenda / Legend: 3, 4, 5 = vertebrae thoracicae 3, 4, 5, C = costa completae spuria, cc = capitulum costae, fiv = fossa iliaca ventralis, fr = fossa renalis, proct = processus transversus, tc = tuberculum costae, VC1, VC2, VC3, VC4, VC5 = vertebra caudalis libera 1, 2, 3, 4, 5. Tabela 9: Meritve sokrižnice (synsacrum) in medenice (pelvis) (v cm) z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 9: Measurements of the synsacrum (cm), with mean (x) and standard deviation (SD). Živali Specimen Celotna dolžina Total length Dolžina medenice Pelvis length Dolžina vzdolž sinsakralnih vretenc Synsacrum vetebrae length Najmanjša širina Minimal breadth Širina v sredini Middle breadth 1 9,4 9 9,6 1,8 4,0 2 9,2 8,9 7,4 1,7 3,6 3 10,5 9,1 7,8 1,6 3,4 4 11,5 9,8 8,5 1,8 4,0 x 10,2 9,2 8,3 1,7 3,8 SD 1,1 0,4 1,0 0,1 0,3 Golob et al.: Anatomske značilnosti skeleta sive čaplje 71 Kosti medeničnih okončin (ossa membri pelvici) Stegnenica (femur) je razmeroma kratka, saj pri odrasli živali meri okrog 9,0 cm (Tab. 10 in 15), goleničnonartna kost (os tibiotarsus) pa okrog 20,8 cm (Tab. 11 in 15). Mečnica (fibula) je v poprečju dolga 9,7 cm (razpon od 9,0 do 10,2 cm) in sega približno do sredine goleničnonartne kosti. Nartnostopalna kost (tarsometatarsus) meri v dolžino povprečno 15,3 cm, največja širina proksimalnega konca meri povprečno 1,4 cm, najmanjša širina telesa 0,6 cm in največja širina distalnega konca 1,4 cm (Tab. 12). Distalni konec nartnostopalne kosti ima tri ločene in izrazite sklepne površine v obliki valja (trochleae metatarsi II, III etIV). Meritve prstnic zadnje okončine so prikazane v tabelah 13 in 14. Prvi prst (hallux) je obrnjen nazaj; meri 5,6 cm v dolžino in ima dve prstnici. Drugi prst (digitus secundus) meri okrog 8 cm in ima tri prstnice, tretji prst (digitus tertius) meri 10,5 cm in je najdaljši, četrti prst (digitus quartus) ima pet prstnic in je dolg 8,6 cm. Drugi, tretji in četrti prst so obrnjeni naprej (anizodaktilni tip noge). Payne in Risley (1976) sta predstavila osnovne značilnosti skeleta čapelj (Ardeidae) in jih primerjala med vrstami te družine, kakor tudi z nekaterimi drugimi skupinami (Balaenicipitidae, Scopidae, Ciconiidae, Threskiornithidae, Phoenicopteridae). Med proučenimi omenjata tudi dva skeleta sive čaplje (Ardea cinerea), ki pa v rezultatih nista prikazana. Našim rezultatom so primerljivi podatki, ki sta jih navedla za ameriško sivo čapljo (Ardea herodias; 12 skeletov). To je skladno s splošno sprejetim stališčem, da sta siva čaplja (Ardea cinerea) in ameriška siva čaplja (Ardea herodias) v tesnem sorodstvu in sta si podobni. Kellner-jeva (1986) je proučila postkranialni skelet evropskih čapelj (Ardea cinerea, Ardea purpurea, Casmerodius albus, Egretta garzetta, Ardeola ralloides, Bubulcus ibis, Nycticorax nycticorax, Ixobrychus minutus, Botaurus stel-laris). Opisala in izmerila je prsnico (sternum), krokarnico (os coracoideum), plečnico (scapula), vilice (furcula), nadlahtnico (humerus), koželjnico (radius), komolčnico (ulna), zapestnodlančnico (carpmetacarpus), medenico (pelvis), stegnenico (femur), goleničnonartno kost (tibiotarsus) in nartostopalnico (tarsometatarsus). Ugotovila je trend sekundarnega spolnega dimorfizma, samci imajo nekoliko krepkejše kosti, vendar pa te razlike niso statistično značilne. Iz njenih podatkov za sivo čapljo smo izračunali poprečne vrednosti za vse živali, ki jih je izmerila (tj. za samce, samice in živali z neugotovljenim spolom) in dobili vrednosti, ki se ne razlikujejo od naših rezultatov oz. so razlike neznatne (Tab. 15). Poleg meritev omenjenih kosti smo v naši raziskavi proučili še morfometrične parametre lobanje ter prstnic in prstov. Tabela 10: Meritve stegnenice (femur; v cm): posebej za levo in desno stegnenico ter za obe skupaj (L+D); z aritmetičnimi sredinami (x) in standardnimi odkloni (SD). Table 10: Measurements of the femur (cm), with mean (x) and standard deviation (SD). L-left, D-right femur. Največja proks. Najmanjša širina Največja distalna Največja distal. Žival Dolžina Length širina Maximal proximal telesa Minimal corpus širina Maximal distal globina Maximal distal Specimen breadth breadth breadth depth L D L D L D L D L D 1 9,4 9,4 1,6 1,6 0,7 0,7 1,6 1,6 1,4 1,4 2 8,9 8,8 1,5 1,5 0,7 0,6 1,5 1,5 1,4 1,3 3 8,5 8,5 1,3 1,4 0,8 0,8 1,2 1,2 1,2 1,1 4 9,2 9,2 1,6 1,7 0,8 0,8 1,5 1,5 1,3 1,3 X 9,0 9,0 1,5 1,6 0,8 0,7 1,5 1,5 1,3 1,3 SD 0,4 0,4 0,1 0,1 0,1 0,1 0,2 0,2 0,1 0,1 L+D 9,0 ± 0,4 1,5 ± 0,1 0,7 ± 0,1 1,5 ± 0,2 1,3 ± 0,1 72 Acta Biologica Slovenica, 59 (1), 2016 Tabela 11: Meritve goleničnonartne kosti (tibiotarsus; v cm), leve in desne ter obeh skupaj (L+D). Table 11: Measurements of the tibiotarsus (cm), with mean (x) and standard deviation (SD). L-left, D-right tibiotarsus. Žival Celotna dolžina Najmanjša širina telesa Globina distal. konca Specimen Total length Minimal corpus breadth Distal end depth L D L D L D 1 21,5 21,3 0,6 0,5 1,5 1,5 2 20,2 20,4 0,5 0,5 1,3 1,3 3 19,0 19,1 0,5 0,5 1,3 1,3 4 22,3 22,2 0,5 0,6 1,4 1,4 x 20,8 20,8 0,5 0,5 1,4 1,4 SD 1,5 1,3 0,1 0,1 0,1 0,1 L+D 20,8 ± 1,3 0,5 ± 0,05 1,4 ± 0,1 Tabela 12: Meritve nartostopalnice (tarsometatarsus; v cm): posebej leve in desne ter obeh (L + D). Table 12: Measurements of the tarsometatarsus (cm), with mean (x) and standard deviation (SD). L-left, D-right tarsometatsarsus. Žival Specimen Celotna dolžina Total length Največja proks. širina Maximal proximal breadth Najmanjša širina telesa Minimal corpus breadth Največja distalna širina Maximal distal breadth L D L D L D L D 1 15,8 15,8 1,5 1,5 0,6 0,6 1,5 1,5 2 14,8 14,8 1,3 1,4 0,5 0,6 1,3 1,3 3 14,1 14,0 1,2 1,3 0,6 0,5 1,3 1,3 4 16,7 16,7 1,4 1,5 0,6 0,5 1,4 1,4 x 15,4 15,3 1,4 1,4 0,6 0,6 1,4 1,4 SD 1,1 1,2 0,1 0,1 0,1 0,1 0,1 0,1 L+D 15,3 ± 1,1 1,4 ± 0,1 0,6 ± 0,1 1,4 ± 0,1 Tabela 13: Dolžina posameznih prstnic (v cm) pri vseh štirih prstih (x±SD), leve in desne okončine; n = 4. Table 13: Measurements of the pedal phalanges (cm), with mean (x) and standard deviation (SD); n = 4. L-left, D-right phalanges. J | Prst I Prst II Prst III Prst IV | J Digit I Digit II Digit III Digit IV L D L D L D L D 1 3,6 ± 0,2 3,6 ± 0,2 3,7 ± 0,2 3,7 ± 0,1 3,3 ± 0,1 3,3 ± 0,2 2,3 ± 0,2 2,3 ± 0,2 2 2,0 ± 0,3 2,0 ± 0,3 2,8 ± 0,2 2,8 ± 0,2 3,4 ± 0,2 3,4 ± 0,3 2,0 ± 0,2 1,9 ± 0,2 3 1,6 ± 0,3 1,6 ± 0,3 2,4 ± 0,4 2,4 ± 0,3 1,7 ± 0,1 1,7 ± 0,2 4 1,5 ± 0,3 1,5 ± 0,3 1,5 ± 0,2 1,6 ± 0,3 5 1,2 ± 0,2 1,1 ± 03 Golob et al.: Anatomske značilnosti skeleta sive čaplje 73 Tabela 14: Skupna dolžina prstnic (v cm) pri posameznem prstu in izračunana povprečna vrednost za vse proučene živali (n = 4) (x, SD). Table 14: Total length of the digits (cm), with mean (x) and standard deviation (SD). L-left, D-right digits. Žival Prst I Digit I Prst II Digit II Prst III Digit III Prst IV Digit IV Specimen L D L D L D L D 1 6,2 6,2 8,7 8,6 11,5 11,7 9,4 9,6 2 5,3 5,3 7,5 7,5 9,9 10,0 8,4 8,3 3 5,3 5,4 7,5 7,4 9,6 9,6 7,5 7,3 4 5,7 5,7 8,4 8,3 11,2 10,9 9,3 9,0 x 5,6 5,7 8,0 8,0 10,6 10,5 8,7 8,6 SD 0,4 0,4 0,6 0,6 0,9 0,9 0,9 1,0 L + D 5,6 ± 0,4 8,0 ± 0,6 10,6 ± 0,9 8,6 ± 0,9 Tabela 15: Primerjava rezultatov naših meritev (v cm) z rezultati Kellner-jeve (1986) za dolžino plečnice (scapula), krokarnice (coracoid), nadlahtnice (humerus), komolčnice (ulna), stegnenice (femur) in golenonart nice (tibiotarsus). Table 15: Comparasion of measurements of this work and Kellner's (1986). Dolžina Dolžina Dolžina Dolžina Dolžina Dolžina plečnice krokarnice nadlahtnice komolčnice stegnenice golenonartnice Scapula length Coracoid Humerus Ulna length Femur length Tibiotarsus length length length Naše delo 8,4 ± 0,4 6,9 ± 0,2 17,6 ± 0,6 20,8 ± 0,5 9,0 ± 0,4 20, 8 ± 1,3 This work Kellner 1986 * 8,6 ± 0,4 6,8 ± 0,3 17,7 ± 0,6 20,9 ± 0,8 9,1 ± 0,3 21,1 ± 1,1 * Preračunano iz srednjih vrednosti skupin Kellner-jeve (samci, samice, neznan spol) in števila živali v posamezni skupini. * Calculated from the mean values of the Kellner's groups (males, females, not determined sex) and number of animals in each group. Povzetek Predstavljen je skelet sive čaplje (Ardea cinerea), in to nekatere kosti lobanje, vretenca v področju vratu, prsi in sinsakruma, plečni in medenični obroč ter prsnica. Za sivo čapljo je značilen dolg kljun, opremljen z nazobčenim tomi-jem, izrazita kraniofacialna upogibna cona (zona flexoria craniofacialis), dolga čelnica z značilno depresijo (depressio frontalis) v nadočesnem področju in izrazit postorbitalni lok, ki kavdalno prehaja v kratek postorbitalni podaljšek (processus postorbitalis). Podrobneje je opisan kompleks zadnjega dela mandibule, kvadratne kosti in krilne kosti ter njihovih sklepnih področij. Za sivo čapljo so značilni dolg parasfenoidni ročaj (rostrum parasphenoidale), obsežna senčnična kotanja (fossa temporalis) in dodatna plitka podsenčnična kotanja (fossa subtemporalis). V vratnem delu hrbtenice imajo poseben pomen nosač, okretač, šesto vretence, skupina vretenc od 12-tega do 16-tega in zadnje vratno vretence. Šesto vretence in omenjena skupina vretenc (tj. od 12-tega do 16-tega) so močneje kalcificirana od ostalih. Zadnje vratno in prva tri prsna vretenca so v svojem distalnem delu tesneje medsebojno speta, medtem ko so v dorzalnem področju bolj prosta. Področje notarija torej ni iz zraščenih vretenc, manjkata tudi dorzalni (crista dorsalis) in lateralni (lamina transversa notarii) koščeni greben. Pač pa je izrazita koščena zraščenost sinsakruma in kolčnic. Na mestu šiva med črevnico in sinsakrumom v postac-etabularnem področju se nahaja čvrsta sinostozna 74 Acta Biologica Slovenica, 59 (1), 2016 zrast (synostosis iliosynsacralis), ki se grebenasto dviga nad površino. Ključnici sta v apofiznem področju koščeno zraščeni v vilice furcula), ki tvorijo koščen sklep z vrhom gredlja (apex carinae). Krokarnici se v področju prsnice deloma prekrižata, desna je umeščena nekoliko navzpred in ventralno od leve, in oblikujeta vsaka svoj sklep s prsnico (sulcus articularis coracoideus). Na kranialnem koncu držaja prsnice (manubrium s. rostrum sterni) se nahaja izrazit stransko sploščen izrastek (spina externa), medtem ko notranji izrastek manjka inje na njegovem mestu kratek žleb. Na notranji ustni (labrum internum) sta sklepni površini za sinovialni sklep s krokarnicama, obsežnejše sklepno področje pa predstavljata krokarnična žleba. V delu so prikazane še nekatere druge značilnosti skeleta sive čaplje in v preglednicah rezultati meritev posameznih kosti. Summary Skeleton of the grey heron (Ardea cinerea) has been presented, particularly with regard to certain skull bones, vertebrae of the neck, thorax and synsacrum, pectoral girdle and pelvic girdle bones, and sternum. The grey heron's skull is characterized by a long rostrum equipped with the serrated tomium, pronounced craniofacial flexion zone (zona flexoria craniofacialis), long frontal bone with depression (depressio frontalis) and distinct postorbital arch (arcus postorbitalis), which caudally proceeds into a short postorbital process. A complex of the caudal part of mandibula, quadrate bone and pterygoid bone, and of their joint domains is described in detail. The long parasphenoid rostrum (rostrum parasphenoidale), large temporal fossa and shallow subtemporal fossa are characteristic for the grey heron. In the cervical part of the spine, the first, second, sixth and last vertebrae, as well as the region from the twelfth to sixteenth vertebrae, are especially significant. The sixth vertebra and the mentioned group of vertebrae (from 12th to 16th cervical vertebrae) are more intensively calcified than others. The last cervical vertebra and the first three thoracic vertebrae are tightly connected in the ventral part and loosen in the dorsal part. Therefore, the notarium region is not composed of the fused vertebrae and the dorsal and lateral osseous crests (crista dorsalis et lamina transversa notarii) are also missing. However, there is a clear osseous fusion between the synsacrum and hip bone. On the suture place between ilium and synsacrum in the postacetabular region is a firm fusion (synostosis iliosynsacralis) elevating over surface and forming a crest. The clavicles are fused into the fork (furcula) and their apophysis forms a synostosis with the sternal keel apex (apex carinae). The coracoids end at the sternum, each in its own groove (sulcus articularis coracoideus); the right coracoid is placed partly ahead and ventrally to the left coracoid. At the cranial end of the rostrum sterni expressive flattened process (spina externa) is found, however, the internal process (spina interna) is missing, and a short groove is in its place. At labrium internum the joint facets for synovial joints with coracoids are placed, however, widely extended joint area belongs to the coracoid joint grooves (sulci articularis coracoidei). Also some other characteristics of the grey heron skeleton and measurements of individual bones are presented. Zahvala Zahvaljujemo se Mojci Jernejc Kodrič iz Prirodoslovnega muzeja Slovenije za izposojo skeletov sive čaplje. Lidiji Smolar, u.d.i.a. se zahvaljujemo za digitalno obdelavo in grafično opremo slik. Zahvaljujemo se Veterinarski postaji Slovenj Gradec za izdelavo digitalnih rentgenskih slik in pomoč pri njihovi interpretaciji. Za konstruktivne pripombe pri pripravi rokopisa se zahvaljujemo recenzentkama. 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Handbook of the birds of Europe, the Middle East and North Africa: The birds of the Western Paleartic. Oxford University Press. Oxford. Fürbringer, M., 1888. Untersuchungen zur Morphologie und Systematik der Vögel zugleich ein Beitrag zur Anatomie der Stütz und Bewegungsorgane. II Allgemeiner Amsterdam: Theil. Verlag von Holkema. Fürbringer, M., 1902. Zur vergleichenden Anatomie des Brustschulterapparates und der Schultermuskeln. V. Teil. Vögel. Abdruck aus der Jenaischen Zeitschrift für Naturwissenschaft 36 (N. F. 28), 289-736. Gill, F., DonskerD. (Eds.), 2016. IOC WorldBirdList (v 6.2). Preneseno iz: http://www.worldbirdnames. org/, pridobljeno 18.4.2016. Golob, Z., 2011. Funkcionalna anatomija ptičev z osnovami ornitologije. - Fakulteta za naravoslovje in matematiko, Maribor. Janžekovič, F., Zajc T., Klenovšek T., Bavdek S., Golob Z., 2015. Osteologija ptičev: prispevek k slovenskemu anatomskemu izrazju. Acta Biologica Slovenica 58 (2) 77-92. Kellner, M., 1986. Vergleichend morphologische Untersuchungen an Eizelknochen des postkranialen Skeletts in Europa vorkommender Ardeidae. Inaugural - Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig - Maximilians - Universität München. München. King, A.S., McLelland, J., 1984. Birds, Their Structure and Function. 2nd ed. Baillie're Tindall. London. Livezey, B.C., Zusi, R.L., 2006. Higher-order phylogeny of modern birds (Theropoda, Aves: Neornithes) based on comparative anatomy: I. - Methods and characters. Bulletin of the Carnegie Museum of Natural History 37: 1-556. NAV, 2012. Nomina Anatomica Veterinaria. Fifth Ed. (revised version). Published by the Editorial Committee Hannover (Germany), Columbia, MO (U.S.A.), Ghent (Belgium), Sapporo (Japan). Preneseno iz: www.wava-amav.org/Downloads/nav_2012.pdf , pridobljeno 18.4.2016. Pascotto, M.C., Donatelli, R.J., 2003. Cranial osteology in Momotidae (Aves: Coraciiformes). J. Morphol. 258: 32-48. Payne, R.B., Risley, C.J., 1976. Systematics and evolutionary relationships among the herons (Ardeidae). Ann Arbor, Museum of Zoology. University of Michigan. Rigler, L., 1985. Veterinarski anatomski slovar = Index verborum anatomicorum veterinariorum. Slovenska akademija znanosti in umetnosti. Ljubljana. Rigler, L., 1990. Anatomija domačih živali. Osteologia in syndesmologia : skripta za študente veterinarstva, agronomije in živilske tehnologije. Veterinarska fakulteta. Ljubljana. Zajc, T., 2010. Anatomske in morfometrične lastnosti skeleta sive čaplje, Ardea cinerea (Linaeus, 1758). Diplomska naloga, Fakulteta za naravoslovje in matematiko, Oddelek za biologijo. Maribor. Planktothrix rubescens Učinek kratke izpostavitve elektro-oksidaciji na Planktothrix rubescens Maša Čatera, Tinkara Rozinab, Maja Zupančič Justin1* aArhel Ltd., Pustovrhova 15, 1000 Ljubljana, Slovenia bEnvit Ltd., Vojkova 63, 1000 Ljubljana, Slovenia *correspondence: maja.justin@arhel.si Abstract: Cyanobacterial blooms in fresh water bodies have a serious negative impact on human, animal and environmental health. The impact of electro-oxidation of water containing Planktothrix rubescens in electrolytic cell equipped with diamond electrodes has been tested. The treatment had an immediate effect on cyanobacterial phycocyanin and chlorophyll fluorescence response and total cell biovolume. Cyanobacterial number reduced for 80 % 130 h after a single treatment of 500 mL of a water sample through electrolytic cell with a flow rate 1 L/min. Obtained results indicate a great potential of the method for in-lake cyanobacterial control. Keywords: Electrolytic cell, boron doped diamond electrode, cyanobacterial bloom control, hydroxyl radicals Izvleček: Pojav cianobakterijskega cveta v sladkih vodah negativno vpliva na zdravje ljudi, na živali in okolje. Z elektrolitsko celico, opremljeno z borom dopiranima diamantima elektrodama, smo povzročili elektrooksidacijo in testirali njen vpliv na Planktotrix rubescens. Tretiranje je imelo takojšen vpliv na fluorescenco klorofila, na fluorescenco cianobakterijskega fikocianina in na celotni biovolumen cianobakterijskih celic. Volumen vzorca je bil 500 ml, pretok skozi elektrolitsko celico pa je znašal 1l/ min. 130 ur po tretiranju se je število cianobakterijskih celic zmanjšalo za 80 %. Rezultati kažejo na velik potencial te metode pri nadzoru cianobakterijske populacije. Ključne besede: elektrolitska celica, z borom dopirana diamantna elektroda, cianobakterijski cvet, hidroksilni radikali Introduction Increased cyanobacterial concentrations and the resulting toxin formation have negative ecological, biogeochemical, health-related and economic impacts (Paerl et al. 2011). A toxic cyanobacterial species Planktothrix rubescens inhabits deep lakes and can bloom throughout the entire year (Sedmak et. al. 2008). A variety of conventional water treatments (chlorination, coagulation, filtration) have been developed to minimize the harmful effects of cyanobacterial bloom, which are based on the removal of the cyanobacterial biomass and do not completely remove microcystins from water (Falconer et al. 1989, Anderson et al., 2009, Pantelic et al. 2013). 78 Acta Biologica Slovenica, 59 (1), 2016 Introduction of advanced oxidation technologies (ozonation, photochemical degradation, Fenton processes, sonolysis) has made total removal of cyanotoxins possible (de la Cruz et al. 2011, Barrington et al. 2013). The inactivation of cyano-bacteria by electrochemical oxidation (ECO) in electrolytic cell equipped with high-performance electrodes represents a perspective alternative for water treatment with great potential, being economical, environmentally friendly and offering higher treatment efficiency (Zhank et al. 2009). Boron-doped diamond anode (BDDA) is known to have the highest potential of forming hydroxyl radicals (OH) and exceptional chemical inertness and durability. Produced OH are powerful non-selective oxidizing agents, capable to react with organic matter. The aim of our work was to test the effect of the method for the reduction of P. rubescens biomass from natural water sample. Materials and methods The ECO treatment was performed in electrolytic cell, equipped with two 60 cm2 large BDD electrodes (Condias, Germany), serving as anode and cathode, placed parallel 2 mm apart and forming 12 ml treatment chamber. The effect of the ECO was studied on P rubescens taken from Lake Bled, Slovenia. The complete water sample (500 ml) with P. rubescens starting biovolume of 56 mm3/L was pumped through the electrolytic cell at a constant flow rate of 1 L/min. The achieved ECO time on the electrodes was 0.72 s using 3 A current intensity. Submersible Sensors (Cyclops 7, Turner Designs, USA) were used for detecting the change in fluorescence of chlorophyll a (CHL) and phycocyanin (PC) after exposed stress in electrolytic cell. The effect of ECO was also monitored by determination of cyanobacterial biovolume (CEN EN 15204 2006) and the extraction of CHL (ISO 10260 2001) before and immediately after the treatment, and then every 24 hours for 5 days. Results Treating P. rubescens with BDD resulted in immediate effect in PC and CHL fluorescence (Fig. 1, results for CHL not shown). After a transient increase of the PC and CHL fluorescence, they both reduced up to 75 % in treated sample 130 h after the ECO compared to the control. Extraction of CHL supported these results, as its concentration dropped for 80 % (not shown). ECO also affected the cyanobacterial biovolume. It reduced for 21 % immediately after the treatment and for 80 % 130 h later. Figure 1: Phycocyanin fluorescence (line) and cyanobacterial biovolume (bar) in treated and control samples after the electro-oxidation treatment. Slika 1: Fluorescenca fikocianina (črti) in biovolumen cianobakterij (stolpci) v kontrolnih in tretiranih vzorcih po elektro-oksidacijskem tretiranju. Čater et al.: Electro-chemical treatment affects cyanobacterial population 79 Discussion The three selected monitoring approaches enabled us to follow immediate and postponed effects of ECO treatment on P. rubescens. Besides an immediate biovolume reduction and therefore a die-off of a part of the population, a transient increase of fluorescence of PC and CHL immediately after the treatment was observed. This can be contributed to the stress response of the survived part of the cells, where the photosynthetic apparatus was damaged. After Zilinskas and Glick (1981), the increased fluorescence is the result of decoupling of PS II reaction centers from CHL, to which the energy from PC can no longer be transferred. The damages were detrimental; resulting in greatly reduced PC and CHL signals below the control level after 4 days (Fig. 1) and were in positive correlation with low concentration of extracted CHL and biovolume, showing on 80 % reduction of the P rubescens population. Although the ECO time was very short, the immediate and postponed effects of ECO were observed even though OH radicals are known to be short-lived (Pryor 1986). This indicates on gradual decrease of the cyanobacterial population, which is beneficial also from the point of cyanotoxins release. The use of BBD electrode has already proved to be effective also in extracellular cyanotoxins degradation and inactivation (Meglic et al. 2016). The effect of ECO using BDD electrodes on degradation of cyanotoxins in natural water samples is planned to be closely followed in our future experiments, as well as the effect of ECO on other biota for which larger scale experiments are planned. Conclusions Short-term exposure of natural water sample to ECO treatment applying BDD electrodes in laboratory environment caused a visible stress References response of P. rubescens, resulting in an evident decrease in biovolume and concentration of CHL, indicating on efficiency of the novel method as an in-lake cyanobacterial control method. Measurement of the fluorescence of cyanobacterial pigments enabled the quantification and probably also the determination of their physiological state. Performance of simultaneous on-line fluorescence measurements immediately after the ECO treatment are therefore suggested in the case of further activities in natural environment to promptly react on increased density of cyanobacteria or their regrowth after the treatment and to keep the natural biodiversity balance. Povzetek Cvetenje cianobakterij v sladkovodnih telesih predstavlja resno težavo za zdravje ljudi in živali ter za okolje. Testirali smo vpliv okolju prijazne metode, elektro-oksidacije z diamantnimi elektrodami, na številčnost cianobakterij. Uporabili smo vodo iz Blejskega jezera, ki je vsebovala P. rubescens. Pri tretiranju smo 500 ml vzorca vode spustili skozi elektrolitsko celico z delovnim volumnom 12 ml in tokom 3 A. S pretokom 1 L/min smo dosegli elektrooksidacijski čas 0,72 s. Učinek tretiranja na fluorescenčni signal fikocianina in klorofila ter na skupni celični biovolumen je bil takojšen. Fluorescenca in koncentracija klorofila sta 130 ur po tretiranju upadli za 75 in 80 %, prav tako biovolumen cianobakterij. Rezultati kažejo, da ima uporabljena metoda velik potencial za izvajanje kontrole nad cianobakterijami v jezerih. Acknowledgements The study was performed within the project Stop CyanoBloom (LIFE12/ENV/SI/000783). Anderson, D. M., 2009. Approaches to monitoring, control and management of harmful algal blooms (HABs). Ocean and Coastal Management, 52, 342-347. Barrington, D. J., Reichwaldt, E. S., Ghadouani, A., 2013. The use of hydrogen peroxide to remove cyanobacteria and microcystins from waste stabilization ponds and hypereutrophic systems. Ecological Engineering, 50, 86-94. 80 Acta Biologica Slovenica, 59 (1), 2016 CEN EN 15204, 2006. Water quality - Guidance standard on the enumeration of phytoplankton using inverted microscopy (Utermohl technique), 42 pp. de la Cruz, A. A., Antoniou, M. G., Pelaez, M., Hiskia, A., Song, W., O'Shea, K. E., He, X., Dionysiou, D. D., 2011. Can we effectively degrade microcystins? - implications for impact on human health status. Anti-Cancer Agents in Medicinal Chemistry, 11 (1), 19-37. Falconer, I.R. 1989. Effects on human health of some toxic cyanobacteria (blue-green algae) in reservoirs, lakes and rivers. Toxicity assessment, 4, 175-184. ISO 10 260, 1992. Water quality-measurement of biochemical parameters-spectrometric determination of the chlorophyll-a concentration. International Organization for Standardization, Geneva, Switzerland. Meglic, A., Pecman, A., Rozina, T., Lestan, D., Sedmak, B., 2016. Electrochemical inactivation of cyanobacteria and microcystin degradation using a boron-doped diamond anode - A potential tool for cyanobacterial bloom control. Journal of Environmental Sciences, doi:10.1016/j.jes. (2.6.2016) Paerl, H.W., Hall, N.S., Calandrino, E., 2011. Controlling harmful cyanobacterial blooms in a world experiencing anthropogenic and climatic-induced change. Science of the Total Environment, 40, 1739-1745. Pantelic, D., Svircev, Z., Simeunovic, J., Vidovic, M., Trajkovic, I., 2013. Cyanotoxins: Characteristics, production and degradation routes in drinking water treatment with reference to the situation in Serbia. Chemosphere, 91, 421-441. Pryor, W.A. 1986. Oxy-radicals and related species: their formation, lifetimes and reactions. Annual Revew of Physiology, 48, 657-667. Sedmak, B., Elersek, T., Grach-Pogrebinsky, O., Carmeli, S., Sever, N., Lah, T.T., 2008. A survey of ecotoxicologically relevant cyclic peptides from Planktothrix rubescens bloom. Radiology and Oncology, 42, 102-113. Zhang, C., Fu, D., Gu, Z., 2009. Degradation of microcystin-RR using boron-doped diamond electrode. Journal of Hazardous Materials, 172, 847-853. Zilinskas, B.A., Glick, R.E., 1981. Noncovalent intermolecular forces in phycobilisomes of Porphy-ridium cruentum. Plant Physiology, 68 (2), 447-452. 81 INSTRUCTIONS FOR AUTHORS 1. 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Acta Biologica Slovenica (2016) - Vol. 59: št. 1 IN MEMORIAM: Janez MULEC, Gorazd KOSI, Tina ELERSEK: In memoriam Mihael Bricelj (1946-2016).........3 ČLANKI - ARTICLES: Mateja GRAŠIČ, Sabina ANŽLOVAR, Simona STRGULC KRAJŠEK: Germination rate of stinkwort (Dittrichia graveolens) and false yellowhead (D. viscosa) in relation to salinity..................5 Jasna DOLENC KOCE: The effects of leaf extracts of crack willow (Salix fragilis) on the growth of Japanese knotweed (Fallopia japonica)...............................................................................................13 Urban ŠILC, Marija GREGORI: Control of alpine dock (Rumex alpinus) by non-chemical methods.................................................................................................................................................23 Pavlina SKARKOVA, Tea ROMIH, Monika KOS, Sara NOVAK, Veno KONONENKO, Anita JEMEC, Milada VAVROVA, Damjana DROBNE: Gold nanoparticles do not induce adverse effects on terrestrial isopods Porcellio scaber after 14-day exposure.....................................33 Anita JEMEC, Tamara MILIVOJEVIČ, Damjana DROBNE, Kristina SEPČIČ, Janko BOŽIČ, Gordana GLAVAN: No chronic effects on biochemical biomarkers, feeding and survival of carnolian honeybees (Apis mellifera carnica) after exposure to nanosized carbon black and titanium dioxide.....45 Zlatko GOLOB, Srdan BAVDEK, Tina ZAJC, Franc JANŽEKOVIČ, Tina KLENOVŠEK: Nekatere anatomske značilnosti skeleta sive čaplje, Ardea cinerea / Some anatomical characteristics of the skeleton of grey heron, Ardea cinerea........................................................................................55 KRATKI PRISPEVKI - BRIEF COMUNICATION: Maša ČATER, Tinkara ROZINA, Maja ZUPANČIČ JUSTIN: Effect of short exposure to electro-oxidation treatment on Planktothrix rubescens................................................................. 77