ACTA BIOLOGICA SLOVENICA LJUBLJANA 2013 Vol. 56, [t. 1: 3–9 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2013 Vol. 56, [t. 1: 3–9 Achenes of common ragweed (Ambrosia artemisiifolia) in packages of sunflowerachenes foroutdoorbirds Plodovi pelinolistne žvrklje (Ambrosia artemisiifolia) v paketih soncnicnih plodov za prostoživece ptice Simona Strgulc Krajšeka*, Marko Novakb aUniversity of Ljubljana, Biotechnical Faculty, Department of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia bGimnazija Bežigrad, Periceva 4, SI-1000 Ljubljana, Slovenia *correspondence: simona.strgulc@bf.uni-lj.si Abstract:Common ragweed (Ambrosia artemisiifolia) is a highly invasive annual plant, introduced to Europe from North America. One of the important spreading vectors of Ambrosia is trading with contaminated sunflower achenes. We analysed the content of ragweed achenes in the packages of sunflower achenes for outdoor birds, which are available in Slovenian shops and markets. The ragweed achenes were found in 29% of the 28 analysed packages. In three packages, the mass percentage of Ambrosia achenes per kg of animal feed exceeded the maximum content (50 mg/kg) allowed by EU law. The analysis showed that it is not possible to infer the presence or absence of Ambrosia achenes in a sample from the amount of impurities in the package of sunflower achenes for bird feed. Keywords:Ambrosia artemisiifolia, Helianthus annuus,invasivespecies,ragweed, Slovenia, sunflower fruits Izvlecek: Pelinolistna žvrklja (Ambrosia artemisiifolia) je invazivna tujerodna enoletnica, ki je bila v Evropo prinesena iz Severne Amerike. Pomemben vektor širjenja vrste predstavlja transport in trgovanje s plodovi soncnic, ki vsebujejo prime- šane plodove žvrklje. Vraziskavi smo preverjali prisotnost plodov žvrklje v paketih soncnicnih plodov za hranjenje prostoživecih ptic. Plodovi žvrklje so bili prisotni v 29 % od 28 paketov hrane za ptice, ki smo jih kupili v slovenskih trgovinah. V treh paketih je kolicina plodov presegala najvecjo doloceno kolicino plodov žvrklje, kot jo doloca evropska zakonodaja (najvec 50 mg/kg). Ugotovili smo tudi, da kolicina necistoc v paketu ni povezana s prisotnostjo plodov žvrklje. Kljucnebeside:Ambrosia artemisiifolia, Helianthus annuus,invazivnatujerodna vrsta, pelinolistna žvrklja, Slovenija, soncnicni plodovi Acta Biologica Slovenica, 56 (1), 2013 Introduction Common ragweed (Ambrosia artemisiifolia) is an annual plant from the family Asteraceae.Itwas introduced to Europe from North America in the 19th century (Csontos et al. 2010, Hegi 1979). In Europe it is considered an invasive alien species. Itpopulatesfields(corn,sunflower,pumpkin…), riverbanks,anddisturbedplacessuchasrailways and road edges (Hegi 1979). It is detrimental to human health as it produces highly allergenic pollen, which causes late summer hay fever (Kofol Seliger 2001, Taramarcaz 2005). The first records of the presence of Ambrosia artemisiifolia inSloveniaarefromLeskovecnear Krškoin1950(Wraber1983).Innextyearssome newlocalitieswerediscovered,andWraber(1983) marked this species as casual in Slovene flora, dis- persedwithhumanactivities.Thefirstdataabout naturalisation of the species are from Prekmurje (Wraber 1983). In the last years the species has becomepresentinalmostalllowlandsinSlovenia, anditisnowknownasoneofmostinvasivealien plant species in Slovene flora (Jogan and Vreš 1998, Jogan 2001, Jogan et al. 2009). Ambrosia artemisiifolia is an annual plant, 0.2–2.5 m high. One or more male flowers are grouped in numerous much reduced capitula, which include one or more male flowers. Male capitula are grouped in racemes at the end of branches, while female flowers are located at the bases of upper leaves. Ambrosia artemisiifolia is ashort-dayplant,anditsfloweringtimebeginsin late summer (Allard 1943). Its fruits are achenes, each containing one seed. That is the reason for the simplified naming of ragweed achenes as seeds in non-botanical general use (newspapers, media …). Achenes usually drop off the mother plant,andtheybecomepartofsoilseedbank,but theymayalsobedispersedbywaterorbyhuman activities. Seeds have primary and secondary dormancy (Baskin and Baskin 1980), and can remain viable for 40 years (Darlington 1922, Telewski and Zeevaart 2002). One of the important spreading vectors of Ambrosia istradingwithcontaminatedsunflower seeds (more precisely: fruits or achenes) (Jehlik and Hejny 1974, Wraber 1983, Essl et al. 2009). Researchers in Austria have noticed that Ambrosia became frequent in habitats associated with bird feeding during the period 1950–1979 (Essl et al. 2009). In the international European research performedinyears2005to2009,whichinvolved institutes from Switzerland, Germany, Denmark and Slovenia, Ambrosia achenes were found in a large proportion of the analysed sources of bird feed (Frick et al. 2011). The spread of Ambrosia in Slovenia is controlledbytwolegallyadoptedregulations. Thefirstis CommissionRegulation(EU)No574/2011of16 June 2011, concerned with undesirable contents of animal feed, including Ambrosia seeds(Anon. 2011). The limit of intervention is 50 mg of Ambrosia seeds per kg of animal feed (Anon. 2011). The second is the Slovene regulation “Odredba o ukrepih za zatiranje škodljivih rastlin iz rodu Ambrosia” (Decree on measures to suppress harmful plants of genus Ambrosia), that obliges every citizen to remove all Ambrosia plants from hisproperty,andhehastopreventtheplantsfrom developing flowers and fruits (Anon. 2010). The aim of our research was to check if the packages of sunflower seeds for outdoor birds available in Slovene shops and markets contain ragweed achenes, to assess the extent of this contamination and to compare it with legally adopted regulations. Materials and methods In autumn of 2011 we collected 35 samples ofpackagesofsunflowerseedsforbirdfeedfrom variousshopsandmarketsinSloveniaforanalyses of ragweed contamination. The samples were planned to cover: (1) different parts of Slovenia, (2) different shops and suppliers, and (3) differ- entpackagesizes.Everypackagewasnumbered, and the bills from the shops were stored together with the package to keep the information about the date and locality of the purchase. The following data have been collected from packagelabels:packageID,commercialname,size (weight), serial number or LOTnumber, country of sunflower fruit production, date of packaging, type of sunflower nuts (black, white, striped, mixed), and data about the purchase: location of shop, shop name, and date. Thefirststepinseparatingtheragweedachenes from sunflower achenes was hand-sieving. For Strgulc Krajšek and Novak: Ambrosia artemisiifolia in bird feed this purpose, a special sieve with 1.5–2.3 mm wire mesh size was made. Prior to examination ofthesamples,thesievewastestedwithartificial (prepared) mixture of sunflower and ragweed achenes. Since no ragweed achenes stayed on thesieve,andonlyafewsunfloweracheneswere smaller than the mesh size, we considered the hand-made sieve suitable enough for fractioning the samples. Secondly, the small fraction that presumably contained the Ambrosia acheneswasweightedand storedinaplasticbag.Thefractionwithsunflower achenes was weighted as well. Asmall fraction was further analysed using a stereomicroscope (20 × magnification) to sort out ragweed achenes. Achenes were weighted, counted and stored in a small plastic bag. There are two other species from genus Ambrosia present in Slovenia: A. psilostachya and A. trifida, but A. artemisiifolia is the most common one (Buttenschřn et al. 2008–09, Jogan 2001). There is a minor possibility that some of theragweedfruitswefoundinbirdfeedbelonged to other Ambrosia species, since no detailed species determinationwas made based on Ambrosia achenesproperties.Accordingtothefrequencyof all Ambrosia species in Europe and Slovenia, we assumedthatthemajorityofachenesbelongedto Ambrosia artemisiifolia. Results 35 packages of sunflower seeds for bird food were collected in different Slovene shops. Seven packageswereexcludedfromtheanalysisbecause it was not possible to isolate ragweed achenes by using the described methods. Altogether, 2 packages of 5 kg, 24 packages of 1 kg, one package of 0.6 kg, and one package of 0.5 kg of bird feed were analysed (Table 1). We included 13 different producers or importing companies of bird feed, but it is possible that theyhadthesamesourceofrawmaterial.Thedata about the origin of the sunflowers were absent from the majority of the packages labels. They were written only on 8 packages (23%). TheresultsoftheanalysisareshowninTable 2. Ragweed achenes were found in 8 packages of bird feed (29%). In three packages, the mass percentage of Ambrosia seeds per kg of animal feed exceeded the maximum content (50 mg) allowed by EU law (Anon. 2011). Producers of these three packages were Agrolit, Semenarna Ljubljana and AnimaPet. We have included more than one sample of bird feed from the same producer (Erdtmanns, Agrolit, Tukano, and Semenarna Ljubljana), and the same series of raw material in our analysis (Table 3). It is apparent, that there are big differ- encesincontentsofragweedachenesinpackages withinthesamplesofthesameproducer,andeven withinthesameseriesoftherawmaterial(0or35 ragweed achenes in 1 kg of bird feed in the case of Semenarna Ljubljana, 105-PC, Table 3). The analysis of correlation between the numberofragweedachenesandthecontentofthe small fraction (impurities) in the sample showed that there is no clear connection between these parameters. Therefore we cannot infer the presence or absence of Ambrosia seeds in a sample justfromtheamountofimpuritiesinthepackage of sunflower achenes for bird feed. Discussion Ragweed achenes were found in one third of the 28 analysed packages of bird feed. In three packages, the mass percentage of Ambrosia seeds per kg of animal feed exceeded the maximum content(50mg)allowedbyEUlaw(Anon.2011). Thus, these packages should not be sold. From a biological point of view, all packages containing one or more seeds of Ambrosia are problematic. Only one successfully germinated seed that develops into a flowering and afterwards fruiting plant is enough to establish a new population of Ambrosia. Although a smaller number of the seedsreducesthepossibilityofsuchevent,itstill makes it possible. The National Veterinary Institute conducted a similar investigation in 2007/08 and 2008/09, analysing10samplesofbirdfeedeachyear(Frick et al. 2011). Ambrosia acheneswerefoundin7and 5 samples, respectively, with the highest amount of 292 Ambrosia achenes per kg of bird feed. In our samples from 2011, the contamination with Ambrosia acheneswaslower(33%ofcontaminated samples with maximum 198 Ambrosia achenes Acta Biologica Slovenica, 56 (1), 2013 Table 1: Information about the packages of sunflower fruits for bird feed used in the analysis. Packages no.: 2, 6, 7, 10, 11, 19 and 22 (grey shading) were excluded from the analysis (explanation in the text). × – no data, *unpacked sunflower achenes with no commercial name bought on a food market. Tabela 1:Informacije o paketih soncnicnih plodov za hranjenje zunanjih ptic, ki smo jih zbrali za analizo. Pakete 2, 6, 7, 10, 11, 19 in 22 (siva podlaga) smo iz analize izkljucili (razlaga v besedilu). × – ni podatkov, *nepakirani soncnicni plodovi kupljeni na tržnici. Sample Commercial name Producer/Importing Lot/Series Package Countryoforigin number company size (kg) of sunflowers 1 Sonnenblumen Kerne Erdtmanns 210910 1 x 2 Bonami Semenarna Ljubljana 105-PC 1 x 3 Bela soncnicna semena Versele_Laga B-9800 0.6 Belgium 4 Soncnice pisane Roko 480-43486 1 Hungary 5 Hobby Vit Tukano 21300040 1 Bulgaria 6 Menu Complete Friskies EAN 0.4 x 7 Lojna pogaca Vogel pick 21305065 0.2 x 8 Soncnicna semena Tukano 21300042 1 Bulgaria 9 Bonami, Soncnice pisane Semenarna Ljubljana 460-PC 5 x za pticjo hrano 10 Bonami, Hrana za zunanje Semenarna Ljubljana P1330 5 x ptice 11 Bonami, Mešanica semen Semenarna Ljubljana 70507015 1 x 12 Bonami, Semena soncnic Semenarna Ljubljana 460-PC 1 x 13 Bonami, Soncnice Mešane Semenarna Ljubljana P1279 1 x 14 Hobby Vit Tukano M1/17:21:29 1 x /10/10/11 15 Soncnicna semena Roko 480-43486 1 x 16 Bonami Semenarna Ljubljana 105-PC 1 x 17 Soncnice Mešane Semenarna Ljubljana P1278 5 x 18 Soncnice pisane Zoo hoby Škofja Loka 0013 1 x 19 Hobby Vit Tukano 11246 1 x 20 Sonnenblumen Kerne Tierell 772013 1 x 21 Bonami, Mešanica semen Semenarna Ljubljana P1331 1 x 22 Bonami, Semena soncnic Semenarna Ljubljana 460-PC 1 x 23 Soncnicna semena Agrolit 19. 9. 2011 1 x 24 Bonami, Semena soncnic Semenarna Ljubljana 105-PC 1 x 25 Hobby Vit Tukano 21300042 1 Bulgaria 26 Soncnicna semena Roko 11/210 1 Hungary 27 Soncnicna semena Agrolit 4. 12. 2010 1 EU 28 Soncnicna semena Agrolit 19. 9. 2011 1 29 Sonnenblumen Kerne Erdtmanns 210910 1 30 Sonnenblumen Kerne Erdtmanns 210910 1 31 Futter Boy Erdtmanns 210829 0.5 32 Hesa Saaten Tierell CHA70 1 x 33 Soncnicna semena Hobby program 21300042 1 34 Soncnicna semena 1kg AnimaPet 598.10 1 EU 35 x* food market x 1 Strgulc Krajšek and Novak: Ambrosia artemisiifolia in bird feed Table 2: Presence of ragweed achenes in the analysed packages of the bird feed. Tabela 2:Prisotnost plodov žvrklje v pregledanih paketih pticje hrane. Sample number Content of ragweed Number of ragweed Mass % of the small achenes (mg/kg) achenes fraction (including ragweed) 24 198 35 1.9 23 61 11 0.5 34 51 9 0.7 28 45 8 0.5 29 34 6 0.3 14 6 1 0.3 22 6 1 0.3 17 1 1 0.1 1 0 0 0.3 3 0 0 0.4 4 0 0 0.2 5 0 0 6.8 8 0 0 0.4 9 0 0 0.1 12 0 0 0.3 13 0 0 0.5 15 0 0 0.2 16 0 0 0.4 18 0 0 0.2 20 0 0 0.4 25 0 0 0.4 26 0 0 0.2 27 0 0 2.2 30 0 0 0.3 31 0 0 0.7 32 0 0 0.4 33 0 0 0.7 35 0 0 0.4 perkgofbirdfeed),whichmayindicateagradual decrease of the contamination level in bird feed available on Slovene market. InSlovenia,everycitizenisobligedtoremove all Ambrosia plants from his property and he has to prevent the plants from developing flowers (because of the allergenic pollen) and fruits (to limit their dispersal) (Anon. 2010). Therefore it is very important to inform people that it is highly possible to introduce Ambrosia into gardens with contaminated bird feed. In Slovenia this is already in progress. Informations about Ambrosia and the related problems were already published in public journals (Jogan 2011, Bacic 2012,StrgulcKrajšek2013),projectpublications (for example: Buttenschřn et al. 2008–09), and ondifferentwebsites(forexample:ProjectThuja: http://www.tujerodne-vrste.info, DOPPS: http:// www.ptice.si). Povzetek Pelinolistnažvrklja(Ambrosia artemisiifolia) je ena najbolj razširjenih invazivnih tujerodnih rastlinskih vrst na obmocju Slovenije (Jogan et al. 2001). Gre za severnoameriško vrsto, ki je bila v 19. stoletju nehote prinesena v Evropo in Acta Biologica Slovenica, 56 (1), 2013 Table 3: Differences in number of ragweed achenes among the packages of bird feed of the same producer, or the same series of the raw material. Tabela 3:Razlike v prisotnosti plodov žvrklje med paketi istega proizvajalca in iste serije paketov soncnicnih plodov. Producer, Series Sample Package Mass of small fraction Number of ragweed number size (kg) (including ragweed) (g) achenes 1 1 2.9 0 Erdtmanns, 210910 29 1 3.2 6 30 1 3.0 0 Agrolit, 23 1 5.5 11 19. 9. 2011 28 1 5.0 8 33 1 6.8 0 Tukano, 21300042 25 1 3.6 0 8 1 4.4 0 Semenarna Ljubljana, 24 1 18.9 35 105-PC 16 1 3.9 0 Semenarna Ljubljana, 460-PC 9 22 12 5 1 1 4.8 3.1 3.5 0 1 0 seširipredvsemobželeznicah,cestah,ruderalnih mestih in poljih (Jogan 2009). Problematicna je zaradi alergenega peloda, ki povzroca poznopoletni seneni nahod. Eden od pomembnejših vektorjev razširjanja je prenos plodov žvrklje skupaj s soncnicnimi plodovi, ki jih prodajajo za hranjenje prostoživecih ptic (Essl et al. 2009, Frick et al. 2011). V okviru raziskave smo pregledali pakete soncnicnih plodov za hranjenje ptic, ki so naprodaj v trgovinah v Sloveniji. Naš namen je bil ugotoviti, v kolikšni meri se v njih pojavljajo plodovi(oreški)pelinolistnežvrklje.Paketehrane zapticesmonakupilijesenileta2011.Zbralismo 35 paketov, od katerih jih 7 nismo pregledali, ker se z izbranimi metodami iz njih ni dalo lociti plodov žvrklje. Za izolacijo plodov žvrklje smo uporabili metodi sejanja skozi sito in rocnega prebiranjavzorca.Izmed28pregledanihpaketov pticje hrane, smo plodove žvrklje našli v 8 paketih, kar predstavlja29 % pregledanih vzorcev. V treh paketih je kolicina plodov žvrklje presegala zakonsko predpisano zgornjo dovoljeno mejo masnegadeležaplodovpelinolistnežvrklje(Anon. 2011),karustreza9plodovompelinolistnežvrklje na kilogram pticje krme. Ugotovili smo veliko raznolikost vsebnosti plodov žvrklje med paketi istega proizvajalca (uvoznika) in celo znotraj iste serijepakiranjasoncnicnihplodov,karjeverjetno povezano z nacinom pakiranja plodov. Kolicina necistoc, ki jih predstavlja drobna frakcija po sejanju vzorca skozi sito, v paketu ni povezana s kolicino plodov žvrklje. Naslovenskemtržišcusotorejvprodajipaketi soncnicnih plodov za hranjenje zunanjih ptic, ki vsebujejo plodove žvrklje, celo s kolicinami, ki presegajozakonskodolocenozgornjomejo.Nujno je obvešcanje potrošnikov o tem problemu, saj lahko zaradi priljubljenega zimskega hranjenja ptic posamezniki nehote razširjajo to alergeno rastlino in jo celo zanesejo na svoje vrtove, od koder pa jo morajood v skladu z odredbo iz leta 2010 odstranjevati (Anon. 2010). Acknowledgements We would like to thank Metka Škornik from Gimnazija Bežigrad for technical support, and Tinka Bacic for critical reading of the manuscript. Strgulc Krajšek and Novak: Ambrosia artemisiifolia in bird feed References Allard, H.A., 1943. The North American Ragweeds and Their Occurrence in Other Parts of the World. Science, New Series, 98/2544, 292–294. Anon., 2010. Odredba o ukrepih za zatiranje škodljivih rastlin iz rodu Ambrosia.ULRS007-336/2010. http://www.uradni-list.si/1/objava.jsp?urlid=201063&stevilka=3572 (9. 1. 2013). Anon., 2011. Commission Regulation (EU) No 574/2011 of 16 June 2011amending Annex I to Directive 2002/32/EC of the European Parliament and of the Council as regards maximum levels for nitrite, melamine, Ambrosia spp. and carry-over of certain coccidiostats and histomonostats and consolidatingAnnexes I and II thereto. http://eur-ex.europa.eu/LexUriServ/LexUriServ.do? uri=OJ:L:2011:159:0007:0024:SL:PDF. (18. 1. 2013). Bacic, T., 2012. Pelinolistna žvrklja (Ambrosia artemisifolia): tujerodna in invazivna. Trdoživ 1 (2), 4–5. Baskin, J.M., Baskin, C.C., 1980. Ecophysiology of Secondary Dormancy in Seeds of Ambrosia Artemisiifolia. Ecology, 61 (3), 475–481. Buttenschřn, R.M., Waldispühl, S., Bohren, C., Simoncic, A, Lešnik, M., Leskovšek, R., 2008–09. Navodila za zatiranje in preprecevanje širjenja pelinolistne ambrozije (Ambrosia artemisiifolia). Univerza v Kopenhagnu, 47 pp. (http://www.kis.si/datoteke/file/kis/SLO/Publikacije/drugo/ambrozija_ navodila_euphresco.pdf, 8. 4. 2013). Csontos, P., Vitalos, M., Barina, Z., Kiss, L., 2010. Early distribution and spread of Ambrosia artemisiifolia in Central and Eastern Europe. Bot. Helv., 120, 75–78. Darlington, H.T., 1922. Dr. W. J. Beal’s Seed-Siability Experiment. American Journal of Botany, 9, 266–269. Essl, F., Dullinger, S., Kleinbauer, I., 2009. Changes in the Spatio-Temporal Patterns and Habitat Preferences of Ambrosia artemisiifolia During Its Invasion of Austria. Preslia, 81, 119–133. Frick, G., Boschung, H., Schulz-Schroeder, G., Russ, G., Ujcic-Vrhovnik, I., Jakovac-Strajn, B., Angetter, D., John, I., Jřrgensen, J.S., 2011. Ragweed (Ambrosia sp.) Seeds in Bird Feed. Biotechnology, Agronomy, Society and Environment, 15 (S1), 39–44. Hegi, G., 1979. Ilustrierte Flora von Mitteleuropa. Band VI/3. Verlag Paul Parey, Berlin, Hamburg, pp, 261–262. Jehlik, V., Heiny, S., 1974. Main Migration Routes of Adventitious Plants in Czechoslovakia. Folia Geobotanica & Phytotaxonomica 9 (3), 241–248. Jogan, N., 2009. Pelinolistna žvrklja (ambrozija), Ambrosia artemisiifolia.In:Jogan,N.(ed.):Tujerodne vrste. Informativni listi izbranih vrst. Zavod Symbiosis, Grahovo, pp. 13–15. Jogan, N., 2011. Pelinolistna žvrklja (ambrozija) tudi v Ljubljani vse bolj razširjena. Ljubljana 16 (7), 42. Jogan,N.,Bacic,T.,Frajman,B.,Leskovar-Štamcar,I.,Naglic,D.,Podobnik,A.,Rozman,B.,Strgulc Krajšek, S., Trcak, B., 2001. Gradivo za Atlas flore Slovenije. Materialsfor the Atlas of Flora of Slovenia. Center za kartografijo favne in flore, Miklavž na Dravskem polju, 443 pp. Jogan, N., Vreš, B., 1998. Ambrosia artemisiifolia: Hladnikia, 10, 45–47. Kofol Seliger, A., 2001. Rod ambrozija (žvrklja). Proteus 63 (6), 276-278. Strgulc Krajšek, S., 2013. Razširjanje ambrozije s pticjo hrano. Vzajemnost, 2, 82. Taramarcaz, P., Lambelet, C., Clot, B., Keimer,C., HauserC., 2005. Ragweed(Ambrosia) progression and its health risks: will Switzerland resist this invasion? Swiss Med Wkly, 135, 538–554. Telewski, F.W., Zeevaart, J.A.D., 2002. The 120-Yr Period for Dr. Beal’s Seed Viability Experiment. American Journal of Botany, 89 (8), 1285–1288. Wraber, T., 1983. Širjenje pelinolistne ambrozije v Sloveniji. Proteus 45 (8), 315–316.