1 VOL. 51 [T. 1 LJUBLJANA 2008 ACTA BIOLOGICA SLOVENICA prej/formerly BIOLO[kI VESTNIk ISSN 1408-3671 izdajatelj/publisher UDk 57(497.4) Dru{tvo biologov Slovenije 2 Acta Biologica Slovenica, 51 (1), 2008 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2008 Vol. 51, [t. 1: 1–54 Acta Biologica Slovenica Glasilo Društva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Društvo biologov Slovenije – Biological Society of Slovenia Glavni in odgovorni urednik – Editor in Chief Mihael Jožef Toman, e-mail: mihael.toman@bf.uni-lj.si Tehnični urednik – Managing Editor Branko Vreš, e-mail: branko.vres@gmail.com Uredniški odbor – Editorial Board Matija Gogala (SI), Nada Gogala (SI) Nejc Jogan (SI), Alenka Malej (SI), Franc Janžekovič (SI), Harald Niklfeld (A), Livio Poldini (I), Boris Sket (SI), Robert Zorec (SI), Thomas F. J. Martin (USA), Mark Tester (UK), Gerhard Thiel (D) Naslov uredništva – Adress of Editorial Office Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Oblikovanje – Design Žare Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno – Printed on: 2008 Tisk – Print: Tiskarna Pleško d.o.o., Ljubljana Naklada: 500 izvodov Cena letnika (dve številki): 15 € za posameznike, 42 € za ustanove Številka poslovnega računa pri Ljubljanski banki: 02083-142508/30 Publikacijo je sofinancirala Agencija za Raziskovalno dejavnost Republike Slovenije. Acta Biologica Slovenica je indeksirana v – is indexed in: Biological Abstracts, Zoological records. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2008 Vol. 51, [t. 1: 3–11 Sprejeto (accepted): 27.10.2008 Seasonal changes in the contents of nutrients in five macrophyte species from the lake Velenjsko jezero (Slovenia) Sezonske spremembe vsebnosti hranil v petih vrstah makrofitov iz Velenjskega jezera (Slovenija) Zdenka MAZEJ1*, Mateja GERM2 1 ERICo d.o.o., Environmental Research and Industrial Co-operation, Koroška 58, 3320 Velenje, Slovenia; E-mail address: zdenka.mazej@erico.si (*corresponding author) 2 Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia Abstract. The study was designed to depict the seasonal dynamic in relative abundance of macrophyte species, nutrient availability and their content in macrophytes to assess the capability of different species to store nutrients in nutrient rich lake Velenjsko jezero. The concentrations of total nitrogen and total phosphorus in the lake sediment, water and aboveground biomass of macrophytes (Nuphar luteum, Najas marina, Najas minor, Potamogeton lucens and Potamogeton pectinatus) were measured at three locations monthly from June to September 2004. Seasonal variability in the contents of total phosphorus in macrophyte tissues was high, but all examined species reached similar maximal concentration in the beginning of their growth. Later in the season, concentrations declined to a high degree. Seasonal variability in the contents of total nitrogen was smaller. Floating-leaved species Nuphar luteum was present in a low amounts and contained much more total nitrogen in its above-ground tissues than the submersed species. Be- cause of different species life spans, differences in the content of nutrients among species were very high in particular month. Nuphar luteum, Najas minor and Potamogeton lucens were rarely present in the lake and they contributed less to storing nutrients in their biomass. Potamogeton pectinatus was commonly present in the early summer, but in August Najas marina prevailed by far and its growth had high effect on the concentration of nutrients in sediment and water. Therefore removing of 1 t dry weight of Najas marina biomass from the lake would contribute to removal of 2.7 kg of phosphorus and 28.2 kg of nitrogen from the lake. Key words: lake, total nitrogen, total phosphorus, Nuphar luteum (L.) Sibth et Sm., Najas marina L., Najas minor All., Potamogeton lucens L. and Potamogeton pectinatus L. Izvleček. V pričujoči raziskavi smo spremljali sezonske spremembe v abundanci makrofit- skih vrst ter vsebnosti celotnega dušika in celotnega fosforja v sedimentu, vodi in v makrofitih (Nuphar luteum, Najas marina, Najas minor, Potamogeton lucens in Potamogeton pectinatus) Velenjskega jezera. Sezonska nihanja v vsebnosti TP v rastlinah so bila zelo velika, vendar so vse vrste v začetku svoje rasti dosegale podobne maksimalne vsebnosti TP, nato pa so koncentracije s časom večinoma pri vseh vrstah hitro upadale. Sezonska nihanja v vsebnosti TN v rastlinah so bila manjša. Največja maksimalna koncentracija celotnega dušika je bila izmerjena v nadzemnih tkivih v jezeru redko prisotne submerzne vrste s plavajočimi listi – Nuphar luteum. Maksimalne koncentracije v popolnoma potopljenih makrofitih (Najas marina, Najas minor, Potamogeton pectinatus in Potamogeton lucens) pa so bile zelo podobne. Zaradi različnega življenjskega cikla makrofitskih vrst, so bile sicer razlike med vrstami v določenem mesecu zelo velike. Prisotnost vrst Nuphar luteum, Najas minor in Potamogeton lucens je bila v jezeru redka, zato je bil njihov prispevek k zmanjšanju hranil v sedimentu in vodi jezera majhen. Potamogeton pectinatus je bila s svojo relativno zmerno prisotnostjo prevladujoča vrsta v jezeru spomladi, poleti pa je nad njo 4 Acta Biologica Slovenica, 51 (1), 2008 močno prevladala vrsta Najas marina. Njena razrast je imela večji učinek na zmanjšano koncen- tracijo hranil v sedimentu in v vodi. Odstranitev 1 tone suhe biomase makrofita Najas marina v mesecu avgustu, bi prispevala k odstranitvi 2,7 kg fosforja in 28,2 kg dušika iz jezera. Ključne besede: jezero, celotni dušik, celotni fosfor, Nuphar luteum (L.) Sibth et Sm., Najas marina L., Najas minor All., Potamogeton lucens L. in Potamogeton pectinatus L. Abbreviations: TP – total phosphorus, SRP – soluble reactive phosphate, TN – total nitrogen, Pot pec – Potamogeton pectinatus L., Pot luc – Potamogeton lucens L., Naj mar – Najas marina L., Naj min – Najas minor All., Nup lut – Nuphar luteum (L.) Sibth et Sm. Introduction Velenjsko jezero is more and more popular as a recreational resource, which increases the threat of its rapid eutrophication. The lake offers good conditions for the growth of macrophytes, which allowed for their rapid spread within only a few years. There is strong evidence that nutrient avail- ability plays an important role in controlling the development and abundance of macrophytes (Carr & Chambers 1998). After 1997 the aquatic plant Najas marina L. prevailed in Velenjsko jezero, forming weed beds that covered larger areas in August and September. Najas marina is a summer-annual plant that is highly fertile and produces great quantities of seeds. Its presence in Velenjsko jezero disturbs swimmers, fisheries and other lake users. Aquatic macrophytes are often suggested to be accumulators of contaminants and nutrients in surface waters (Wolverton & mCDonalD 1979). Nutrient absorption in submersed macrophytes occur s both from the water by foliage and from the sediments by root and rhizoid systems. The relative contribution of roots and shoots, at least to N and P uptake, depends on the sediment:water nutrient ratio (Carignan 1982). But Wetzel (2001) emphasized that under most circumstances, even in nutrient-rich waters, roots are the dominant sites of nutrient uptake and assimilation for aquatic plants, although some evidence is contradictory (e.g. sWanepoel & vermaak 1977). Experimental analyses have demonstrated that most rooted submersed angiosperms obtain most of their phosphorus from the interstitial water of the sediments (e.g. Chambers & al. 1989; Carr & Chambers 1998), since the absorbable nutrient concentration is much higher in sediment pore water than in the water column (Carr & Chambers 1998). Because of that, the value of tissue analyses of element concentrations in aquatic macrophytes is suspect as an index of the fertility of the lake water. Nutrient content of the water can be quite unrelated to plant growth of those species having ready access to the abundant nutrient in the sediment. The rooted plants can function as a “pump” of nutrients from the sediment; some of those nutrients can then be lost to the water during both active growth and decomposition (Wetzel 2001). Nutrient concentrations in macrophyte species differ greatly even among closely related species and within the same species from site to site (kufel & kufel 2002; garbey & al. 2004). This can be explained by the fact that nutrient uptake and accumulation in macrophytes does not only depend on the physiological capacity of the species, but also on the nutrient concentration in water and sediment (Carignan 1982; sharDenDu & ambasht 1991; fernánDez-aláez & al. 1999) and on N and P reserves in the plant tissue (taheruzzaman & kushari 1989). In the present study we attempted to depict the seasonal dynamics in relative abundance of macro- phyte species, as well as nutrient availability and content in macrophytes, to assess the capability of different species to store nutrients in nutrient rich lake Velenjsko jezero. For this purpose we carried out a comparative analysis of nitrogen and phosphorus content of macrophyte species: the submersed annual species Najas marina and Najas minor All., the submersed perennial annual species Pota- mogeton lucens L. and Potamogeton pectinatus L. and the floating-leaved perennial species – Nuphar luteum (L.) Sibth et Sm. 5Z. Mazej, M. Germ: Seasonal changes in the contents of nutrients in five macrophyte species … Materials and Methods Description of the site Lake Velenjsko jezero is located in central Slovenia, in the Šalek Valley. It is situated at an alti- tude of 366 m, with a surface area of 135,000 m2 and a maximal depth of 54 m. It is an artificial lake resulting from mining activity. The detailed description is available in Šterbenk (1999) and mazej and epŠek (2005). Presence and abundance of macrophytes The distribution of macrophyte species over the entire littoral was assessed using a boat, depth meter, view box and sampling rake to choose sampling locations. After that, three sampling transects (L1, L2, L3) were chosen for comparative analyses on the south-eastern part of the lake, where macro phyte species richness was greatest. Each transect was 200±2 m in length. These transects were surveyed every month from June to September. Species abundance in each section was evaluated accordin g to kohler (1978) on a five level descriptor scale (1 – very rare, 2 – infrequent, 3 – common, 4 – frequent, 5 – abundant, predominant). Content of nutrients in water, sediment and macrophytes Samples of water, sediment and macrophytes were taken from the three locations every month from June to September 2004. The sediment samples were collected from the top 5 cm of bottom sediments using a grab sampler. Samples of water were collected by hand from a boat by submerging precleaned PE bottles approximately 50 cm beneath the water surface. Plants were collected from the boat with a rake. The contents of total nitrogen (TN), total phosphorus (TP) and soluble reactive phosphate (SRP) in sediment were analysed by the standard methods: ISO 11261:1995, ISO 11263:1995 and ÖNORM L 1088:2005, while TN and TP in the water were determined according to the standard methods ISO 10304-2:1995 and ISO 6878:2004. Above ground tissues of plants were washed carefully in the laboratory to remove sediment and periphyton. The specimens of different macrophyte species were analysed for nutrient content. Plant material was oven-dried at 75 oC to constant weight and ground to a fine powder by milling. TN in plants was determined by the standard method ISO 11261:1995, while TP was determined by EPA Method 3050B Mod Block. Statistical analysis Statistical procedures were performed using the Statistica software package (Statistica for Windows, version 7.0). After verifying the normality and homoscedasticidy of the variables (K-S and Liliefors test for normality), standard one-way analysis of variance (ANOVA; LSD test) was used to check the existence of significant differences between the locations regarding the contents of nutrients in water, sediment and macrophytes. The Spearman correlation coefficients between the content of nutrients in macrophytes and the contents of nutrients in water and sediment were calculated. 6 Acta Biologica Slovenica, 51 (1), 2008 Results Table 1: Abundance of different macrophyte species in Lake Velenjsko jezero in the year 2004 at the three selected transects. Each transect was 200±2 m in length. Five level descriptor scale of abundance was used: 0 – absent, 1 – very rare, 2 – infrequent, 3 – common, 4 – frequent, 5 – abundant, predominant (kohler 1978). Tabela 1: Prisotnost in pogostost makrofitov na treh izbranih transektih Velenjskega jezera od junija do septembra 2004. Dolžina transektov je merila 200±2 m. Abundanca po kohler (1978) temelji na petstopenjski skali: 0 – odsotna, 1 – posamična, 2 – redka, 3 – pogosta, 4 – množična, 5 – prevladujoča). L1 L2 L3 Jun Jul Aug Sep Jun Jul Aug Sep Jun Jul Aug Sep Nup lut 2 2 2 2 0 0 0 0 0 0 0 0 Pot pec 3 3 3 2 0 0 0 0 3 3 2 2 Pot luc 0 0 0 0 1 2 2 1 1 2 2 1 Naj mar 0 1 5 4 0 2 5 3 0 2 5 3 Naj min 0 0 0 0 0 0 2 2 0 1 2 2 The distribution pattern of the species was patchy, no species examined being found at all three chosen locations, with the exception of Najas marina, which in August overgrew almost the whole littoral (Tab. 1). Potamogeton pectinatus, Najas minor and Potamogeton lucens throve at two locations and Nuphar luteum at only one. We detected large seasonal changes in the presence and abundance of macrophyte species at the sampling locations due to their different life histories. The perennial spe- � ��� � ��� � ��� � ��� � ��� � � � � � � � ��� � � � � � � ��� � � � � � � ��� ����������������������������������������������������������������������������������������������������������������� � � �� � �� � � �� � � � �� �� � ! " # � ��� ��� ��$ ��% � ��� ��� ��$ � � �� � �� � & �� � � �� � ! " #� � � �� � �' � �� �� �� � (� # ������� � �� � ������� ������� � �� �� ���� ��� � ��� Figure 1: Total nitrogen (TN) content in above-ground tissue of five species of macrophytes (whole plant homoge- nate) – Nuphar luteum, Potamogeton pectinatus, Potamogeton lucens, Najas minor and Najas marina (in columns). Each value is the mean of three replicates. Lines represent the content of TN in sediment and TN in the water column during the summer growth season. Slika 1: Vsebnost celotnega dušika (TN) v nadzemnih tkivih petih makrofitskih vrst – Nuphar luteum, Pota- mogeton pectinatus, Potamogeton lucens, Najas minor and Najas marina (v stolpcih). Vsaka vrednost je povprečje treh paralelk. Linije predstavljajo vsebnost TN v jezerskem sedimentu in v vodi. 7Z. Mazej, M. Germ: Seasonal changes in the contents of nutrients in five macrophyte species … cies Nuphar luteum, Potamogeton lucens and Potamogeton pectinatus started their vegetative growth earlier in the season. Potamogeton pectinatus prevailed in June and July, while in August Najas marina became the most abundant and overgrew almost all littoral. Annual species Najas marina and Najas minor had short life spans, appearing in the lake from July to September only. The contents of TN, and especially of TP, in plants were higher at the beginning of their develop- ment (Figs. 1–2). Thus the comparison of different species regarding their content of nutrients was influenced by differences in phenological phase. Extreme values of TN stand out, the highest being 4.16% DW in Nuphar luteum in July (Fig. 1). This species was found only at the first location (L1). Concerning TP, the species Nuphar luteum (0.41% DW) and Potamogeton pectinatus (0.37% DW) from the first location (L1) and Najas marina (0.41% DW), Najas minor (0.42% DW) and Potamogeton lucens (0.38% DW) from the second location (L2) contained similar maximal concentrations at the beginning of their development (Fig. 2). With respect to water and sediment, there were no significant differences in chemical characteristics in these two compartments between the three locations, except that the sediment of the first location contained a significantly higher amount of TP than the sediment from the third location (p = 0.015). Accordingly, Potamogeton pectinatus and Najas marina from the first location contained significantly higher average contents of TP than the same species from the third location – (Potamogeton pecti- natus – p = 0.034, n=12 and Najas marina – p = 0.002, n=12). No significant differences were found � ��� ��� ��� ��� ��� � � � � � � ��� � � � � � � ��� � � � � � � ��� ����������������������������������������������������������������������������������������������������������������� � � �� � �� � � �� � � � �� �� � ! " # � � � � � � $ % & ' �� �� � ( � �� � ) �� � * �� � �� � ) �� � � �� �� � +, � #� � � * * �� � �- � �� �� �� � +� # ������� � �� � ������� ������� � �� �� ����������� ��� ������� ��� ���������� ��� Figure 2: Total phosphorus (TP) content in above-ground tissue of five species of macrophyte (whole plant homogenate) – Nuphar luteum, Potamogeton pectinatus, Potamogeton lucens, Najas minor and Najas marina (in columns). Each value is the mean of three replicates. Lines represent the content of soluble reactive phosphorus (SRP) and TP in sediment and TP in the water during the summer growth season. ** Values of TP in sediment were in fact 100 times higher than is represented on the figure. ** Values of TP in water were in fact 100 times lower than is represented on the figure. Slika 2: Vsebnost celotnega fosforja (TP) v nadzemnih tkivih petih makrofitskih vrst – Nuphar luteum, Pota- mogeton pectinatus, Potamogeton lucens, Najas minor and Najas marina (v stolpcih). Vsaka vrednost je povprečje treh paralelk. Linije predstavljajo vsebnost lahko dostopnega fosforja (SRP) in TP v jezerskem sedimentu in TP v vodi. ** Vsebnosti TP v sedimentu so bile 100 krat večje, kot je prikazano na sliki. ** Vsebnosti TP v vodi so bile 100 krat manjše kot je prikazano na sliki. 8 Acta Biologica Slovenica, 51 (1), 2008 between different locations regarding concentrations of TN in macrophytes. Concentrations of SRP in the sediment, and TP and TN in water, drastically decreased in September, while content of TN in sediment remained constant. Table 2: Correlation (Spearman coefficient) between the concentrations of nutrients in some macrophyte species and the concentrations in water and sediment. Tabela 2: Korelacija (Spearmanov koeficient) med koncentracijo hranil v nekaterih makrofitskih vrstah in med koncentracijo hranil v vodi in sedimentu. Annual species Perennial species Naj mar (15) Naj min (15) Pot pec (24) Nup lut (12) Pot luc (24) TN TP TN TP TN TP TN TP TN TP Water (TN, TP) (12) 0.60* 0.42* 0.56* 0.43* –0.33 –0.38**–0.88** –0.15 –0.3 0.23 Sediment (TN, SRP) (12) 0.05* 0.41* –0.4* 0.67* –0.02 0.57* 0.41** –0.47 0.01 –0.33 Level of statistical significance: *** p<0.001, ** p<0.01, * p<0.05 Numbers of replicates are in brackets. Relationship between the content of nutrients in the macrophytes and in water and sediment was determined (Tab. 2). The contents of total phosphorus in Najas marina and Najas minor correlated positively with the contents of SRP and TP in sediment, while their contents of TN correlated positively with that in the water. Negative significant correlation was observed between the content of TN in Nuphar luteum and its content in water. Discussion Lake Velenjsko jezero can be classified as eutrophic on the basis of the level of total phosphorus (0.1 mg/L) and total nitrogen (1.34 mg/L) in the water (OECD 1982). Macrophytes of Velenjsko jezero showed no evidence of TP and TN limitation. The TN and TP contents in macrophytes were generally higher than the critical concentrations of 0.13 % for TP and 1.3 % for TN. The critical concentration of a nutrient in a plant has been defined as that concentration in plant tissues which permits the maximum yield (gerloff & krombholz 1966). The variation in macronutrient concentration in macrophyte tissues was found to correlate with the growth form of the plant (jaCkson & kalff 1993; fernánDez-aláez & al. 1999). The macroalgae showed the lowest nutrient content, while an impoverishment in N was observed in emergent species (fernánDez-aláez & al. 1999). The lower N in tissues of emergent plants as related to submersed and rooted floating-leaved macrophytes is explained by their greater biomass, which means a greater proportion of non-nitrogenou supporting tissue (fernánDez-aláez & al. 1999). The above-ground tissues of Nuphar luteum contained much more TN than those of submersed species, in spite of the fact that the structure of floating leaves is similar to that of leaves of emergent species. Floating-leaved plants have a morphological adaptation to increase inorganic carbon and mineral acquisition, which enables their higher productivity (boston & al. 1989). The floating and submersed leaves of Nuphar luteum enable it to exploit CO2 from air and from water. Like emergent macrophytes, Nuphar luteum possesses operationally active transpiration-mediated root-pressure systems, which enable nutrient absorption and translocation from the roots to the foliage (Wetzel 2001). Maximum concentration of TP in Nuphar luteum did not differed from those of submersed species. furtaDo (1998) registered the changes in concentrations of both nutrients (P and N) as a function of season, plant age and stage of vegetative development. Our results show great seasonal variability in the content of nutrients in macrophyte tissues in which fluctuations of the content of TP were most marked (Figs. 1–2). Seasonal variability in the contents of total phosphorus in macrophyte tissues 9Z. Mazej, M. Germ: Seasonal changes in the contents of nutrients in five macrophyte species … was high, but all examined species reached similar maximum concentration in the beginning of their growth. Later in the season, concentrations declined to a considerable degree. Seasonal variability in the contents of total nitrogen was smaller. The highest content of nutrients, especially phosphorus, at the beginning of the growth season is known as “luxury” uptake. Such a strategy may benefit the plant later, should nutrient concentrations diminish (garbey & al. 2004), and allow the abundance of submerged plants to increase (palma-silva & al. 2002). The rarely present floating-leaved species Nuphar luteum contained much more total nitrogen in its above-ground tissues than submersed species. Because of different species life spans, differences in the content of nutrients among species were very large in particular month. Nuphar luteum, Potamogeton lucens and Potamogeton pectinatus overwinter, and then grow rapidly in the spring. They are able to recycle and withdraw nutrients from their senescing parts or storage organs for reuse (vitousek 1982) and so are less dependent on the nutrient concentration in their environment at the beginning of their growth, other than annual plants. Their growth in the lake was not significantly influenced on nutrient concentration in sediment and water. In contrast annual plants like Najas marina and Najas minor, are more dependent on the concentration of nutrients in their environment and required high levels for new growth in the summer, resulting in intensive growth. In July the soft sediment of the littoral is still not colonized and the amounts of nutrients in the water and sediment do not decrease before September. Both Najas species propagate from seeds that enable quick colonization of new habitats (agami & Weisel 1986). Favourable nutrient conditions in summer offered Najas marina to overgrow almost the whole lake littoral in one month, reflecting the nutrient status of its environment. Concentrations of SRP in the sediment and TP in water drastically decreased in September, after the rapid expansion of N. marina in August. The content of TN in water also decreased drastically in September, while its content in sediment remained constant. Removing of 1 t dry biomass of Najas marina from the lake would contribute to removal of 2.7 kg of phosphorus and 28.2 kg of nitrogen from the lake. Conclusions 1. Floating-leaved species Nuphar luteum contained much more total nitrogen in its above-ground tissues than submersed species. 2. Macrophytes showed great seasonal variability of TP content in their tissues. The stage of plant vegetative development appears to be an important factor, influencing the content of nutrients in plants. A high content of TP, very similar at all species, was detected in the initial phase of plant development. Because of different species life spans, differences in the content of nutrients among species were high in particular months. 3. While the development of four other species did not significantly influenced the nutrient concen- tration in sediment and water, rapid development of annual Najas marina had a significant effect on the concentration of nutrients in these two compartments. Therefore removal of at least part of the enormous biomass of this species in August would contribute to a high export of nutrients from Velenjsko jezero. Acknowledgements The authors are grateful to Prof. Dr. Alenka Gaberščik and Prof. Roger Pain for critical reading of the manuscript. This research was a part of the project financed by the Šoštanj Thermal Power Plant: “The role of macrophytes in maintaining the ecological balance of the Velenje Lake ecosystem” and of the project financed by the Slovenian Research Agency (ARRS), Ljubljana: “Determination of heavy metal pollution in the Šalek lakes by using bioindicators” No. L1-7144-1007. Additional support was 10 Acta Biologica Slovenica, 51 (1), 2008 provided by the research program “Communities, relations and communications in ecosystems” (P1- 0255), financed by Slovenian Research Agency (ARRS), Ljubljana. Parts of analyses were performed in the laboratory at National institute of Biology. References agami m. & Waisel y. 1986: The ecophysiology of roots of submerged vascular plants. – Végétale 24: 607–624. boston, h. l., aDams, m. s., maDsen, j. D. 1989: Photosynthetic strategies and productivity in aquatic systems. Aquatic Botany 34: 27–57. Carignan r. 1982: An empirical model to estimate the relative importance of roots in phosphorus uptake by aquatic macrophytes. Canadian Journal Fisheries and Aquatic Sciences 39: 243–247. Carr g. m. & Chambers p. a. 1998: Macrophyte growth and sediment phosphorus and nitrogen in a Canadian prairie river. Freshwater Biology 39: 525–536. Chambers, p. a., prepas e. e., bothWell m. l. & hamilton h. r. 1989: Roots versus shoots in nutrien t uptake by aquatic macrophytes in flowing waters. Canadian Journal Fisheries and Aquatic Sciences 46: 435–439. fernánDez-aláez m., fernánDez-aláez C. & béCares e. 1999: Nutrient content in macrophytes in Spanish shallow lakes. Hydrobiologia 408/409: 317–326. furtaDo a. l. s. 1998: Ash free dry weight, organic carbon, nitrogen and phosphorus content of Typha domingensis Pers. (Thyphaceae) and aquatic macrophyte. Verhandlungen – Internationale Vereinigung für Limnology 26: 1842–1845. garbey C., murphy k. j., thiébaut g. & muller s. 2004: Variation in P-content in aquatic plant tissues offers an efficient tool for determining plant growth strategies along a resource gradient. Freshwater Biology 49: 346–356. gerloff g. C. & krombholz p. h. 1966: Tissue analysis as a measure of nutrient availability for the growth of angiosperm aquatic plants. Limnology and Oceanography 11: 529–537. International standard ISO 11261:1995. Soil quality – Determination of total nitrogen – Modified Kjeldahl method. Brussels, Belgium. International standard ISO 11263:1995. Soil quality – Determination of phosphorus – Spectrometric determination of phosphorus soluble in sodium hydrogen carbonate solution. Brussels, Belgium. International standard ISO 10304–2: 1995. Water quality – Determination of dissolved anions by liquid chromatography of ions – Part 2: Determination of bromide, chloride, nitrate, nitrite, orthophos- phate and sulfate in waste water. Brussels, Belgium. International standard ISO 6878:2004. Water quality – Determination of phosphorus – Ammonium molybdate spectrometric method. Brussels, Belgium. jaCkson l. j. & kalff j. 1993: Patterns in metal content of submerged aquatic macrophytes: the role of plant growth form. Freshwater Biology 29: 351–359. kohler a. 1978: Methoden der Kartierung von Flora und Vegetation von Süßwasserbiotopen. Land- schaft und Stadt 10: 78–85. kufel l. & kufel i. 2002: Chara beds acting as nutrient sinks in shallow lakes – a review. Aquatic Botany 72: 249–260. mazej z. & m. epŠek 2005. The macrophytes of lake Velenjsko Jezero, Slovenia – the succession of macrophytes after restoration of the lake. Acta Biologica Slovenica 48: 21–31. OECD 1982: Eutrophication of waters, monitoring, assessment and management. Paris. ÖNORM L 1088: 2005. Chemische Bodenuntersuchungen. Bestimmung von pflanzenverfugbarem Phosphat und Kalium nach der Doppel-Lactat (DL)-Methode. Wien, Austria. 11Z. Mazej, M. Germ: Seasonal changes in the contents of nutrients in five macrophyte species … palma-silva C., albertoni e. f. & estevas f. a. 2002: Clear water associated with biomass and nutrient variation during the growth of a Charophyte stand after a drawdown, in a tropical coastal lagoon. Hydrobiologia 482: 79–87. sharDenDu & ambasht r. s. 1991: Relationship of nutrient in water with biomass and nutrient accu- mulation of submerged macrophytes of a tropical wetland. New Phytology 117: 493–500. Šterbenk e., 1999. Šalek Lakes. Pozoj Velenje. sWanepoel j. h. & vermaak j. f. 1977: Preliminary results on the uptake and release of 32P by Pota- mogeton pectinatus. Journal of the Limnological Society of Southern Africa 3: 63–65. taheruzzaman Q. & kushari D. p. 1989: Evaluation of some common aquatic macrophytes cultivated in enriched water as possible source of protein and biogas. Hydrobiologia 23: 207–212. vitousek p. m. 1982: Nutrient cycling and nutrient use efficiency. American Naturalist 119: 553–572. Wetzel r. 2001: Limnology, 3rd edition. Academic Press, New York, USA. Wolverton b. C. & mCDonalD r. C. 1979: Upgrading facultative wastewater lagoons with vascular aquatic plants. J. Water Poll. Control Fed. 51: 305–313. ACTA BIOLOGICA SLOVENICA LJUBLJANA 2008 Vol. 51, [t. 1: 13–20 Sprejeto (accepted): 27.10.2008 Competitive advantages of Najas marina L. in a process of littoral colonization in the lake Velenjsko jezero (Slovenija) Tekmovalne prednosti vrste Najas marina L. pri kolonizaciji litorala Velenjskega jezera (Slovenia) Zdenka MAZEJ1*, Mateja GERM2 1 ERICo d.o.o., Environmental Research and Industrial Co-operation, Koroška 58, 3320 Velenje, Slovenia; E-mail address: zdenka.mazej@erico.si (*corresponding author) 2 Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia Abstract. Najas marina is the dominant macrophyte species in Velenjsko jezero. It appeared in the lake in 1997 and soon prevailed over the species Myriophyllum spicatum L. and Pota- mogeton crispus L., which used to be the two most abundant species in the lake in the past. The physico-chemical and geomorphological characteristics of the lake are discussed in relation to the attributes of Najas marina, presenting competitive advantages in this environment. Conditions in the lake such as warm water and unstable sediment enabled successful growth and life strategy of Najas marina, which is a summer-annual plant with short life cycle, quick propagation from seeds and a very extensive root system. Key words: artificial lake, physico-chemical and geomorphological characteristics of the lake, plant invasion, aquatic macrophytes, Najas marina L. Izvleček. Najas marina je prevladujoča makrofitska vrsta v Velenjskem jezeru. V jezeru se je pojavila leta 1997 in kmalu prevladala nad vrstama Myriophyllum spicatum L. in Potamogeton crispus L., ki sta do takrat prevladovali v jezeru. V članku so izpostavljene fizikalno kemijske in geomorfološke značilnosti jezera, ki so ene izmed pomembnih dejavnikov, ki vplivajo na kompeticijske lastnosti določene vrste. Razmere v jezeru, kot so topla voda in nestabilen sediment, omogočajo uspešen razvoj in rast vrste Najas marina, ki je toploljubna enoletnica s kratkim življenjskim ciklom, hitrim načinom razmnoževanja iz semen in z zelo ekstenzivnim koreninskim sistemom. Ključne besede: umetno jezero, fizikalno kemijske in geomorfološke značilnosti jezera, invazivnost rastlin, vodni makrofiti, Najas marina L. Introduction The ability of a plant species to invade a region depends not only on the attributes of the plant, but also on the physico-chemical characteristics of the lake habitat invaded (ali & soltan 2006). Besides penetration of radiation and temperature, the key factor determining the composition and vertical distribution of submersed macrophyte communities is substrate composition (average grain size, sorting level, silt fraction, organic matter). Sediments influence vegetation in two ways, i.e. by serving as an anchor for roots and rhizoids (hanDley & Davy 2002) and as potential nutrient reservoir 14 Acta Biologica Slovenica, 51 (1), 2008 (peralta & al. 2003). sCulthorpe (1967) pointed out that the principal influence of the substrate on the distribution of rooted aquatic plants is due to its physical texture rather than chemical composition. The sediment texture is a very important characteristic, which has very great influence on the physical, chemical and, indirectly, on the biological properties of the sediment. The mechanical properties and instability of the substrate in aquatic habitats might prevent seedling establishment and increase the chances of dislodgement (titus & hoover 1991) in many species. In contrast seedlings of Najas marina L. can not colonize on firm sediments (hanDley & Davy 2002). Authors who have investigated the interaction between vegetation and sediment (linDner 1978, selig & al. 2007) clearly distinguished between plant communities’ characteristic for mineral sediments with low nutrient content and plant communities’ characteristic for muddy sediments with high nutrient content. Najas marina is a rare species in Slovenia, registered in only six locations (Vreš & Kaligarič 1999, germ & al. 2008) beside Velenjsko jezero, where Najas marina is the dominant species. It appeared in the lake in 1997 (mazej 1998) and outcompeted both Myriophyllum spicatum L. and Potamogeton crispus L., which used to be abundant in previous years. However, they had never occurred in the lake to such an extent as Najas marina. Myriophyllum spicatum and Potamogeton crispus are known as very competitive species, which establish large monospecific weed beds in many lakes (niChols & shoW 1986; bolDuan & al. 1994), including some Slovenian meso-eutrophic lakes (mazej 1998; Mazej & gaberščiK 1999). We hypothesize that physico-chemical and geomorphological characteristics of the lake are the main factors, which allow the broad expansion of Najas marina. Species composition was related to environmental characteristics in order to point out the reasons for the successful growth of Najas marina, which prevailed over other macrophytes. Some of the attributes of Najas marina were also stressed. Materials and methods Study Area Velenjsko jezero is situated in the Šalek valley, in the Sub-alpine part of Slovenia near the Aus- trian border at an altitude of 366 m, with a surface area of 135000 m2 and a maximum depth of 54 m. Huge lignite-coal reserves, which are dug in Velenje Colliery are the crucial factor of human caused changes and pollution of the Šalek valley. The most remarkable consequences of coal mining are three subsidence lakes, Škalsko, Velenjsko and Družmirsko jezero. Velenjsko jezero came into existence after the World War II. At the beginning of its existence it was used as a reservoir for ash transport water from the Šoštanj thermal plant. The pH of transport water is around 12. The pH of lake was the same so any sorts of organism could not survive in such an alkaline environment. Up to early eighties ash slurry had run into lake, but afterwards the building of the ash landfill was begun. Ash reminded on the landfill and only transport water ran to lake. The pH remained 12 because the only reason for high alkinity was transport water. The closed loop system for the ash was built in 1994 and this has an impact on the lake quality. In only three years the lake pH has almost been normalized and biota appeared in the lake again (Šterbenk, 1999). It was recolonized by phyto- and zooplankton, fish, macrophytes and other organisms. The pH-value of the lake is now around 8. Sampling Three sampling locations (L1, L2, L3) with highest species richness and similar morphometric characteristics of littoral on the south-eastern part of the lake, were chosen. Samples of water and sedi- ment were taken adjacent to vegetation from three sampling locations monthly from June to September 2004. Samples of water were taken at 0.5 m depth in plastic bottles, while sediment was sampled by a 15Z. Mazej, M. Germ: Competitive advantages of Najas marina L. in a process of littoral colonization … handled plastic scoop. Water transparency was measured in the middle of the lake with a Secchi disk. Temperature at 30 cm and pH at all three locations were measured with a MultiLine P4. Presence and abundance of macrophytes At the same time the distribution of macrophyte species at the three sampling locations was assessed using a boat, a depth meter, a viewing box and a sampling rake. Species abundance was evaluated according to kohler (1978) on a five level descriptor scale (1 – very rare, 2 – infrequent, 3 – common, 4 – frequent, 5 – abundant, predominant). Water and sediment analysis The samples of water and sediment were brought to the laboratory and stored at 4 oC. The contents of total nitrogen (TN), total phosphorus (TP) and soluble reactive phosphate (SRP) in sediment were analysed by the standard methods: ISO 11261:1995, ISO 11263:1995 and ÖNORM L 1088:2005, while TN and TP in the water were determined according to the standard methods ISO 10304-2:1995 and ISO 6878:2004. Soil texture, sorting level and the content of organic matter were also determined in the sedi- ment. Soil texture was determined by mechanical analysis – the sedimentation stactometer method with American classification according to hoDnik (1988). The percentages of four fractions were determined: sand (grain size > 0.2 mm), coarse silt (grain size 0.05 0,05). The influence of different teachers was considered, so all 21 classes were taught and supervised during evaluations by the author, who is a biology teacher by profession. The lesson was of standard duration (45 min). Instrument The tests were administered one week before instruction (pre-test) and three times after instruc- tion (post-tests 1, 2, 3). The first post-test was administered one week, the second two months and the third four months after the lesson. The pre-test consisted of two parts. The first required students to answer two open-ended questions about which animals they are afraid of, and which four they like the most (affection). They also had to explain their answers. The follow-up consisted of 10 questions testing the students’ knowledge about 42 Acta Biologica Slovenica, 51 (1), 2008 amphibians. Seven were multiple choice, two required them to provide missing information, and one required that they identify animals in a picture. The second part of the pre-test included a self-report scale, which required students to rate their attitude toward three amphibian species (green frog, toad and salamander) on a five-point scale and state whether or not they had had any direct experience with these animals (yes/no statements). We had presumed that the students at least knew the species names of the selected animals before the lesson, as they are quite common locally. Students rated their fondness for individual amphibian spe- cies according to the following scale: 1 = ‘I don’t want anything to do with this animal’; 2 = ‘I don’t like this animal’; 3 = ‘I do not have any special feelings toward this animal’; 4 = ‘I like this animal’; 5 = ‘I like this animal very much’. In this part of the questionnaire students also assessed their fear of and disgust toward a selection of 20 animals, which also included the green frog, the toad and the salamander (considered for publica- tion elsewhere). Here students had to list for the second time whether they had any direct experience with amphibians. Students’ answers on prior direct experience with three amphibian species from this section of the questionnaire were used to test the reliability of their answers. Test-retest reliability for the green frog was modest (r = 0,614; p < 0,001). Reliabilities for the toad (r = 0,918; p < 0,001) and the salamander (r = 0,834; p < 0,001) were high. Statistical Analysis Basic descriptive statistics was used to obtain the average values and frequencies of each data component or group. Nonparametric tests were used to determine statistically significant differences between and within the experimental and control groups on attitude ratings and knowledge test scores. All the data was analysed with the SPSS statistical programme, version 15.0.0. Results Direct experience Before the lesson, few pupils stated that they had had previous experience with the selected animals (Tab. 1). In comparison with the experimental group (Exp), more students in the control group (Kla) stated that they had had previous direct experience with the green frog (Chi2 (1, n = 383) = 3,674; p = 0,055). In both groups, it was the toad that the fewest pupils had previous direct contact with (Chi2 (1, n = 377) = 0,231; p = 0,631), while the most knew the salamander first hand (Chi2 (1, n = 379) = 0,775; p = 0,379). Table 1: Descriptive statistics on students’ prior direct experience with amphibians according to type of instruction. Tabela 1: Deskriptivna statistika predhodne neposredne izkušnje učencev z dvoživkami glede na način pouka. ANIMAL Instr. DIRECT EXPERIENCE No yes no answer n f (%) n f (%) n f (%) Green frog Exp 184 69,4 73 27,5 8 3,0 Kla 78 61,4 48 37,8 1 0,8 Toad Exp 205 77,4 48 18,1 12 4,5 Kla 103 81,1 21 16,5 3 2,4 Salamander Exp 146 55,1 110 41,5 9 3,4 Kla 76 59,8 47 37,0 4 3,1 43I. Tomažič: The influence of direct experience on students’ attitudes to, and knowledge about amphibians Direct experience and attitude Before the lesson, there were no statistically significant differences between the two groups (Exp and Kla) in rating their attitudes toward an individual amphibian species. (Mann-Whitney U; all p > 0,01). Fig. 1 shows the average ratings of the students’ attitudes towards the three amphibian species before and after both lessons, i.e. the one that included live animals and the one that did not. The rat- ings are shown in relation to students’ previous direct experience with individual amphibian species. Statistically significant differences occurred only in ratings pertaining to the salamander, with students who had had previous direct experience with the animal. (Figure 1c; Kla1-Exp1; Mann Whitney U, Z = -2,902; p = 0,004). All graphs show that, on average, pupils with no prior direct experience with individual animals rated their attitude lower, which means that it was more negative. In contrast, pupils who had had previous direct experience with the animals on average rated their attitude higher. After the lesson which included the use of live animals (Exp), students with both higher and lower rated initial attitudes (Exp0 and Exp1) significantly changed their attitude toward the animals. In the Exp0 group, the differences between the initial and final ratings for individual animals were statistically significant (Wilcoxon Signed Ranks Test; all p < 0,001). The same holds true for the Exp1 group (Wilcoxon Signed Ranks Test; all p < 0,001). Figure 1: Average students’ attitude ratings for individual amphibian species before and after instruction, according to prior direct experience and instruction type. Chart order: (a) green frog, (b) toad and (c) salamander. Exp0 and Kla0: students without prior direct experience with animals. Exp1 and Kla1: students with prior direct experience with animals. Slika 1: Povprečna ocena odnosa učencev za posamezno vrsto dvoživk pred in po pouku glede na predhodno neposredno izkušnjo in način pouka. Vrstni red: (a) zelena žaba, (b) krastača in (c) močerad. Exp0 in Kla0: učenci brez predhodne neposredne izkušnje z živalmi; Exp1 in Kla1: učenci s predhodno neposredno izkušnjo z živalmi. 44 Acta Biologica Slovenica, 51 (1), 2008 With the control group students who had had prior direct experience with the animals (Kla1), there were no statistically significant differences between their initial and final ratings concerning individual animals (Wilcoxon Signed Ranks Test; all p > 0,05). The rating of their attitude toward the toad (Fig. 1, b) fell somewhat, but not statistically significantly. The lesson which did not include live animals (Kla), on the other hand, had a positive effect on the attitude of pupils without prior direct experience (Kla0) of the animals. Differences in attitudes toward the toad and the green frog between the initial and final ratings in the Kla0 group were statistically significant (Wilcoxon Signed Ranks Test; both p < 0,01), while the attitudes toward the salamander were marginally significant (Wilcoxon Signed Ranks Test; p = 0,055). Direct experience and knowledge Students’ knowledge about amphibians before and after the lesson did not differ significantly between the two groups (Tab. 2). The differences in achievement scores before and after instruction were rather small. Table 2: Descriptive and inference statistics of average students’ test scores before and after instruction, according to type of instruction. Tabela 2: Deskriptivna in inferenčna statistika povprečnih rezultatov testov znanja učencev pred in po pouku glede na način pouka. test Instruction sig. Exp (n = 265) Kla (n = 127) Mean score (%) SD Mean score (%) SD Z p Pre-test 43,4 15,6 43,3 14,5 –0,491 0,623 Post-test 1 63,8 13,1 65,4 13,2 –1,135 0,256 Post-test 2 61,4 14,2 58,1 19,0 –0,798 0,425 Post-test 3 60,7 14,7 57,7 15,8 –1,724 0,085 Note: sig.: Mann-Whitney test. After analysing the data about direct experiences with individual amphibian species before instruction, differences in knowledge between students became evident and statistically significant (Fig. 2). On average, pupils who attended a lesson which did not involve live animals (Kla) scored similarly on the first post-test as the Exp1 pupils. However, knowledge of the Kla0 and Kla1 pupils showed a more rapid decrease in subsequent testing in comparison to the pupils from Exp0 and Exp1 groups. In the first post-test, the Exp0 pupils achieved the lowest scores in the part which tested their knowledge; however, in subsequent tests, their knowledge showed a steadier decline than in the Kla group. In final testing, there were no statistically significant differences between the Kla0, Kla1 and Exp0 groups (Kruskal-Wallis test; all p > 0,05). Pupils who displayed the most knowledge were those who had direct contact with the animals before and during the lesson. Discussion Slovenian science curricula stipulate that at the beginning of their compulsory education students should encounter various living organisms. The results of our survey show that amphibians are rarely among them, as much as 55% or more of students had had no direct contact with individual amphibian species (Tab. 1). Moreover, the results suggest that students seldom encounter live amphibians outside the school environment (e.g. family influence). 45I. Tomažič: The influence of direct experience on students’ attitudes to, and knowledge about amphibians ������ � � �������� � ��������� � ���� � ������ � ������ � ��� � ����� � ����������� � �� � �������� � ��� ����������������������������������������������������������������������� ����������� ����������������������������������� ��!�����"��! ����������#�������������������� ����������������������� ��$�����"��$ ����������#������������������������������� ��������� %��&� �� �'�����(�� � ��)������ � ������ � )���*� � �(����� ����� � �� � �� ����&� �� �����)��� � )� �*���������������������)&�+�*������������)������������,��&��-��������� ����� )������,���������&�����(�����������(������ ��!����"��! ��(��������)����������� ������������)&�+�*��)�,������.� ��$����"��$ ��(���������������������������� �)&�+�*��)�,������� / Figure 2: Average students' test scores before and after instruction in relation to students' prior experience of individual amphibian species. Chart order: (a) green frog, (b) toad and (c) salamander. Exp0 and Kla0: students without prior direct experience of animals. Exp1 and Kla1: students with prior direct experience of animals. Slika 2: Povprečni rezultati testov znanja učencev pred in po pouku v povezavi z njihovimi predhodnimi izkušnjami s posamezno vrsto dvoživk. Vrstni red: (a) zelena žaba, (b) krastača in (c) močerad. Exp0 in Kla0: učenci brez predhodne neposredne izkušnje z živalmi; Exp1 in Kla1: učenci s predhodno neposredno izkušnjo z živalmi. The pedagogical literature suggests several ways in which students could work with amphibians (murphy & fortner 2001, green & green 2005, tomasek & al. 2005). However, there is little infor- mation about how this influences their attitudes toward and knowledge about these animals. The pupils included in our survey who had had prior direct experience of amphibians showed better attitudes toward these animals before the lesson than pupils who had had no such experience (Fig. 1). kellert (1985) makes a similar point in his research, stressing that the focus with young students should be firstly on developing the emotional component of experiencing animals. This stage should be followed by acquiring information about them. Only when students are familiar with animals and have developed appropriate attitudes towards them (experiencing animals through direct contact and acquiring enough factual information) can they, according to Kellert, develop their own understanding of ecology and their ethical concerns for the welfare of animals (aged 13 or older). Pupils who had neither been in contact with amphibians before the lesson nor encountered them during the lesson (Kla0) changed their attitudes more significantly than those who did not encounter amphibians during the lesson, but had previous direct experiences with them (Kla1, Fig. 1). With the Kla0 pupils, the teacher’s role was probably greater, as he presented his own view of and experience with 46 Acta Biologica Slovenica, 51 (1), 2008 the animals. Pupils who had no prior experience (Kla0) found it much easier, through information passed on to them by the teacher, to form a positive attitude towards the animals than the Kla1 pupils. Kla1 pupils’ prior experience with the animals probably affected the change in their attitudes, since an attitude arising from direct experience shows more resistance to external influences (fazio & zanna 1981). The students who gained the most were those who attended the lesson where live animals were used (Exp; Figure 1). Both those who had no prior direct experience (Exp0) and those who had prior direct experience (Exp1) rated their attitudes toward the animals higher than their peers who did not see live animals during the lesson (Kla). We believe that the teacher’s guidance played a vital part in this, as the teacher would notice and immediately correct any false ideas pupils may have had about the animals. After the lesson, the attitude of the Exp0 pupils was at roughly the same level as that of the Exp1 students before the lesson. The latter, however, changed their attitudes so much that, on average, these could be rated as “I like this animal”. The two groups (Exp and Kla) also differed in terms of their knowledge (Fig. 2). The Kla0, Kla1 and Exp0 pupils achieved similar scores in the final test, while the Exp1 group, which had prior direct experience of the animals and worked with live animals during the lesson, achieved a better score. The latter also showed better knowledge retention. One week after the lesson, the Exp0 students showed the weakest knowledge, which decreased evenly by the time of the final test. It is possible that these students scored poorer than the Exp1 group as a result of their emotional involvement during the les- son, for which reason they required more time to switch from emotionally experiencing an animal to becoming rationally involved in the lesson. Although Slovenian curricula maintain that students should develop their relationship toward nature and the living organisms, there are often no clear guidelines about how teachers are supposed to achieve this. In their work teachers focus greatly and spend considerable amounts of time on passing information to their pupils about animals and their endangerment. A question arises here: will students who have never encountered a living organism be prepared to do something for them? linDemann – mathies (2005) finds that students who are in contact with organisms (even the more inconspicuous ones, such as plants) develop more sensitivity toward them. Moreover, they develop a liking for them and quite a special attitude. Furthermore, these students show greater knowledge of these organisms. As strgar (2007) notes, when students work with plants their interest in them increases and even more so after teacher intervention. Our experience about the interest and motivation of students in our survey is almost the same. Students who attended the lesson which used live animals were highly motivated at the end and ready to learn more about them (they did not wish to leave the classroom and they asked many questions). Based on our research, we believe that prior direct experience with animals outside the classroom and relatively short exposure (45min) to live animals in the classroom have a positive effect on pupils’ attitude toward amphibians. If the educational process provided ‘the right’ experiences with living organisms, there would probably be fewer questions raised about potentially harmful behaviour toward animals as noted by barney & al. (2005). Conclusion The results of our research show that: 1. enabling pupils brief direct experience significantly changes their attitude toward living organisms, which helps to reduce their intrapersonal barriers on contact with living organisms, 2. higher grade teachers could compensate for pupils’ ‘forgotten’ or missed opportunities to emotion- ally experience animals in elementary classes, 47I. Tomažič: The influence of direct experience on students’ attitudes to, and knowledge about amphibians 3. students who gained the most knowledge and had the best attitude were those who had prior direct experience with animals and attended the lesson in which live animals were used; therefore, it would be sensible that, during the course of their education, students were given several opportunities to experience the same living organisms (e.g. in elementary school, secondary school). Worth researching in the future would be the attitude of both future teachers and teachers of life science topics toward living organisms, as they have an important role and the responsibility to create such learning environment in which students can further develop their knowledge and attitudes. In addition, it would be beneficial to study the extent to which a teacher’s attitude toward living organisms can affect the attitude of students. The lesson which involved live amphibians would make a good starting point for students’ subse- quent work, as once they were in direct contact with the animals, they also asked questions to which they could later find answers on their own. Povzetek V raziskavi, ki je potekala v šolskem letu 2004/2005, smo preverjali, kako neposredna izkušnja učencev z nekaterimi dvoživkami vpliva na spremembo njihovega odnosa do in znanja o dvoživkah. V raziskavo je bilo vključenih 392 učencev 21 sedmih razredov devetletne osnovne šole. Sodelujoči učenci so bili stari od 11 – 12 let. Po kellertu (1985) je to obdobje, v katerem učenci intenzivno pri- dobivajo podatkovna znanja. To obdobje naj bi sledilo obdobju izgradnje čustvene skrbi in navezanosti otrok do živali. Zato smo raziskavo zasnovali tako, da smo pri učencih preverili njihov odnos do in znanje o dvoživkah pred poukom in izvedli tri zaporedna preverjanja po pouku. Statistična obdelava podatkov je upoštevala predhodne neposredne izkušnje učencev z dvoživkami. Da bi v raziskavi preprečili vpliv različnih učiteljev, je vsa preverjanja in pouk izvedel raziskovalec. Učenci so eno šolsko uro spoznavali dvoživke. Pouk je bil izveden na dva načina. Pri prvem načinu so učenci spoznavali dvoživke preko pogovora, razlage in uporabe terciarnih učnih virov (slikovnega materiala). Ta način pouka je služil kot kontrola. Pri drugem načinu pouka pa smo uporabili žive živali, kjer smo učence spodbujali k vzpostavitvi neposrednega stika z živalmi. Rezultati so pokazali, da večina učencev pred poukom še ni imela neposrednih izkušenj z dvoživkami. Učenci, ki niso imeli predhodne neposredne izkušnje s posamezno živaljo, so živalim v povprečju pripisali nižje ocene odnosa, kar pomeni, da je bil njihov odnos 'slabši' od učencev, ki so pred poukom že imeli neposredno izkušnjo. Po pouku so najboljši odnos izrazili učenci, ki so se pri pouku srečali z živimi živalmi. Pri pouku, kjer žive živali niso bile uporabljene, pa so v manjši meri spremenili odnos samo tisti učenci, ki niso imeli predhod- nih neposrednih izkušenj. Pri slednji skupini učencev je bila verjetno prisotna večja moč informacij, ki jih je posredoval učitelj. Pri zadnjem preverjanju znanja se je izkazalo, da so učenci s predhodno neposredno izkušnjo in uporabljenimi živimi živalmi pri pouku izkazali najvišje znanje. Raziskava je pokazala, da s kratkotrajno neposredno izkušnjo pri učencih pomembno spremenimo njihov odnos do organizmov in tako pozitivno vplivamo na znižanje intrapersonalnih ovir, s katerimi se učenci srečajo ob stiku z organizmi. Z uporabo živali lahko nadoknadimo ˝pozabljeno˝ doživljanje le-teh v nižjih razredih šolanja. Na znanju so največ pridobili in imeli najboljši odnos učenci, ki so imeli predhodne neposredne izkušnje z živalmi in so doživeli živali pri pouku. Zato bi bilo smiselno, da bi se učenci v času šolanja večkrat srečali z istimi skupinami organizmov, začenši že v nižjih razredih šolanja otrok. V nadaljevanju bi bilo pomembno ugotoviti, ali so učitelji in bodoči učitelji usposobljeni za delo z živimi organizmi pri pouku, saj imajo učitelji pomembno vlogo in odgovornost, da ustvarjajo učno okolje, kjer lahko učenci pridobijo največ na znanju in odnosu. 48 Acta Biologica Slovenica, 51 (1), 2008 References barney E. C., J. J. mintzes, & C. F. yen 2005: Assessing Knowledge, Attitudes, and Behavior towar d Charismatic Megafauna: The Case of Dolphins. Journal of Environmental Education 36(2): 41–55. beebee T. J. C. & R. A. griffiths 2005: The amphibian decline crisis: A watershed for conservation biology? Biological Conservation 125(3): 271–285. bixler R. D. & M. F. floyD 1999: Hands On or Hands Off? Disgust Sensitivity and Preference for Environmental Education Activities. Journal of Environmental Education 30(3): 4–11. blaustein A. R. & J. M. kieseCker 2002: Complexity in conservation: lessons from the global decline of amphibian populations. Ecology Letters 5 (4): 597–608. bogner F. X. 1998: The Influence of Short-Term Outdoor Ecology Education on Long-Term Variables of Environmental Perspective. Journal of Environmental Education 29(4): 17–29. Davey G. C. L., K. Cavanagh, & A. lamb 2003. Differential aversive outcome expectancies for high- and low-predation fear-relevant animals. Journal of Behavior Therapy and Experimental Psychiatry 34(2): 117–128. Davey G. C. L., A. S. mCDonalD, U. hirisave, G. G. prabhu, S. iWaWaki, C. I. jim, & al. 1998: A cross-cultural study of animal fears. Behaviour Research and Therapy 36(7–8): 735–750. fazio R. H. & M. P. zanna 1981: Direct experience and attitude-behavior consistency. In: Berkowitz L. (ed.): Advances in experimental social psychology, Vol. 14. Academic Press, San Diego: CA, pp. 161–202. greene J. S. & B. D. greene 2005: Using Amphibians and Reptiles to Learn the Process of Science. Science Activities: Classroom Projects and Curriculum Ideas 41(4): 18–21. kellert S. R. 1985: Attitudes toward animals: age-related development among children. Journal of Environmental Education 16(3): 29–39. kellert S. R. 1996: The value of life: biological diversity and human society. Island Press, Washing- ton: DC, 263 pp. killermann W. 1998: Research into biology teaching methods. Journal of Biological Education 33(1): 4–9. leeming F. C., W. O. Dwyer & B. A. Bracken 1995: Children’s Environmental Attitude and Knowledge Scale: Construction and Validation. Journal of Environmental Education 26(3): 22–31. linDemann-matthies P. 2005: ‘Loveable’ mammals and ‘lifeless’ plants: how children’s interest in common local organisms can be enhanced through observation of nature. International Journal of Science Education 27(6): 655–677. loCk R. 1994: Biology – the study of living things. Journal of Biological Education 28(2): 79–80. loCk R. & P. alDerman 1996: Using animals in secondary school science lessons: Teacher experience and attitude. Journal of Biological Education 30(2): 112–118. looy H. & J. R. WooD 2006: Attitudes toward Invertebrates: Are Educational “Bug Banquets” Effec- tive? Journal of Environmental Education 37(2): 37–48. lukas K. E. & S. R. ross 2005: Zoo Visitor Knowledge and Attitudes toward Gorillas and Chimpan- zees. Journal of Environmental Education 36(4): 33–48. morgan J. M. 1992: A theoretical basis for evaluating wildlife-related education-programs. American Biology Teacher 54(3): 153–157. murphy T. P. & R. W. fortner 2001: The case of the missing anurans. American Biology Teacher 63(9): 670–676. ranDler C., A. ilg, & J. kern, 2005: Cognitive and Emotional Evaluation of an Amphibian Con- servation Program for Elementary School Students. Journal of Environmental Education 37(1): 43–52. 49 ross S. R., K. E. lukas, E. V. lonsDorf, T. S. stoinski, B. hare, R. shumaker & al. 2008: Science priorities – Inappropriate use and portrayal of chimpanzees. Science 319(5869): 1487–1487. strgar J. 2007: Increasing the interest of students in plants. Journal of Biological Education 42(1): 19–23. thompson T. L. & J. J. mintzes 2002: Cognitive structure and the affective domain: on knowing and feeling in biology. International Journal of Science Education 24(6): 645–660. tomasek T. M., C. E. mattheWs & J. hall 2005: What’s slithering around on your school grounds? Transforming student awareness of reptile and amphibian diversity. American Biology Teacher 67(7): 419–425. tomkins S. P. & S. D. tunniCliffe 2001: Looking for ideas: observation, interpretation and hypothesis- making by 12-year-old pupils undertaking science investigations. International Journal of Science Education 23(8): 791–813. yen C. F., T. W. yao & Y. C. Chiu 2004: Alternative Conceptions in Animal Classification Focusing on Amphibians and Reptiles: A Cross-Age Study. International Journal of Science and Mathematics Education 2(2): 159–174. yore L. B. & S. boyer 1997: College students’ attitudes towards living organisms: The influence of experience and knowledge. American Biology Teacher 59(9): 558–563. 51 NAVODILA AVTORJEM 1. Vrste prispevkov a) ZNANSTVENI ČLANEK je celovit opis originalne raziskave in vključuje teoretični pregled tematike, podrobno predstavljene rezultate z diskusijo in sklepe ter literaturni pregled: shema IMRAD (Introduction, Methods, Results And Discussion). Dolžina članka, vključno s tabelami, grafi in slikami, na sme presegati 15 strani; razmak med vrsticami je dvojen. Recenzirata ga dva recenzenta. b) PREGLEDNI ČLANEK objavi revija po posvetu uredniškega odbora z avtorjem. Število strani je lahko večje od 15. c) KRATKA NOTICA je originalni prispevek z različnih bioloških področij (sistematike, biokemije, genetike, mikrobiologije, ekologije itd.), ki ne vsebuje podrobnega teoretičnega pregleda. Njen namen je seznaniti bralca s preliminarnimi ali delnimi rezultati raziskave. Dolžina na sme presegati 5 strani. Recenzira ga en recenzent. d) KONGRESNA VEST seznanja bralce z vsebinami in sklepi pomembnih kongresov in posvetovanj doma in v tujini. e) DRUŠTVENA VEST poroča o delovanju slovenskih bioloških društev. 2. Originalnost prispevka Članek, objavljen v reviji Acta Biologica Slovenica, ne sme biti predhodno objavljen v drugih revijah ali kongresnih knjigah. 3. Jezik Teksti naj bodo pisani v angleškem jeziku, izjemoma v slovenskem, če je tematika zelo lokalna. Kongresne in društvene vesti so praviloma v slovenskem jeziku. 4. Naslov prispevka Naslov (v slovenskem in angleškem jeziku) mora biti kratek, informativen in razumljiv. Za naslovom sledijo imena avtorjev in njihovi polni naslovi (če je mogoče, tudi štev. faxa in e-mail). 5. Izvleček – Abstract Podati mora jedrnato informacijo o namenu, uporabljenih metodah, dobljenih rezultatih in zaključkih. Primerna dolžina za znanstveni članek naj bo približno 250 besed, za kratko notico pa 100 besed. 6. Ključne besede – Keywords Število naj ne presega 10 besed, predstavljati morajo področje raziskave, predstavljene v članku. Člankom v slovenskem jeziku morajo avtorji dodati ključne besede v angleškem jeziku. 7. Uvod Nanašati se mora le na tematiko, ki je predstavljena v članku ali kratki notici. 8. Slike in tabele Tabele in slike (grafi, dendrogrami, risbe, fotografije idr.) naj v članku ne presegajo števila 10, v članku naj bo njihovo mesto nedvoumno označeno. Ves slikovni material naj bo oddan kot fizični original (fotografija ali slika). Tabele in legende naj bodo tipkane na posebnih listih (v tabelah naj bodo le vodoravne črte). Naslove tabel pišemo nad njimi, naslove slik in fotografij pod njimi. Naslovi tabel in slik ter legenda so v slovenskem in angleškem jeziku. Pri citiranju tabel in slik v besedilu uporabljamo okrajšave (npr. Tab. 1 ali Tabs. 1–2, Fig. 1 ali Figs. 1–2; Tab. 1 in Sl. 1). 9. Zakjučki Članek končamo s povzetkom glavnih ugotovitev, ki jih lahko zapišemo tudi po točkah. 52 Acta Biologica Slovenica, 51 (1), 2008 10. Povzetek – Summary Članek, ki je pisan v slovenskem jeziku, mora vsebovati še obširnejši angleški povzetek. Velja tudi obratno. 11. Literatura Uporabljene literaturne vire citiramo med tekstom. Če citiramo enega avtorja, pišemo allan (1995) ali (allan 1995), če sta dva avtorja (trinajstić & Franjić 1994), če je več avtorjev (pullin & al. 1995). Kadar navajamo citat iz večih del hkrati, pišemo (honsig-erlenburg & al. 1992, WarD 1994a, allan 1995, pullin & al. 1995). V primeru, če citiramo več del istega avtorja, objavljenih v enem letu, posamezno delo označimo s črkami a, b, c itd. (WarD 1994a,b). Če navajamo dobesedni citat, označimo dodatno še strani: toman (1992: 5) ali (toman 1992: 5–6). Literaturo uredimo po abecednem redu, začnemo s priimkom prvega avtorja, sledi leto izdaje in naslov članka, mednarodna kratica za revijo (časopis), volumen poudarjeno, številka v oklepaju in strani. Npr.: honsig-erlenburg W., k. krainer, p. milDner & C. Wieser 1992: Zur Flora und Fauna des Webersees. Carinthia II 182/102 (1): 159–173. trinajstić & J. Franjić 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253–256. WarD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10–15. WarD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10–15. Knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo sledeče: allan J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp. pullin a. s., i. f. g. mClean & m. r. Webb 1995: Ecology and Conservation of Lycaena dispar: British and European Perspectives. In: pullin A. S. (ed.): Ecology and Conservation of Butterflies, 1st ed. Chapman & Hall, London, pp. 150–164. toman M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1–7. 12. Format in oblika članka Članek naj bo poslan v obliki Word dokumenta (doc) ali kot obogateno besedilo (rtf) v pisavi “Times New Roman CE 12” z dvojnim medvrstnim razmakom in levo poravnavo ter s 3 cm robovi na A4 formatu. Odstavki naj bodo med seboj ločeni s prazno vrstico. Naslov članka in poglavij naj bodo pisani krepko in v velikosti pisave 14. Vsa latinska imena morajo biti napisana ležeče. Uporabljene nomenklaturne vire navedemo v poglavju Metode. Tabele in slike so posebej priložene tekstu. Vse strani (vključno s tabelami in slikami) morajo biti oštevilčene. Glavnemu uredniku je potrebno oddati original, dve kopiji in elektronski zapis na disketi 3,5”, na CD-romu ali kot priponko elektronske pošte (slednjega odda avtor po opravljenih strokovnih in jezikovnih popravkih). 13. Recenzije Vsak znanstveni članek bosta recenzirala dva recenzenta (en domači in en tuji), kratko notico pa domači re- cenzent. Avtor lahko v spremnem dopisu predlaga tuje recenzente. Recenziran članek, ki bo sprejet v objavo, popravi avtor. Po objavi prejme 30 brezplačnih izvodov. V primeru zavrnitve se originalne materiale vrne avtorju skupaj z negativno odločitvijo glavnega urednika. 53 INSTRUCTIONS FOR AUTHORS 1. Types of Articles a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a theoretical survey of the topic, a detailed presentation of results with discussion and conclusion, and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and Discussion). The length of an article including tables, graphs, and illustrations may not exceed fifteen (15) pages; lines must be double-spaced. Scientific articles shall be subject to peer review by two experts in the field. b) REVIEW ARTICLES will be published in the journal after consultation between the editorial board and the author. Review articles may be longer than fifteen (15) pages. c) BRIEF NOTES are original articles from various biological fields (systematics, biochemistry, genetics, microbiology, ecology, etc.) that do not include a detailed theoretical discussion. Their aim is to acquaint readers with preliminary or partial results of research. They should not be longer than five (5) pages. Brief note articles shall be subject to peer review by one expert in the field. d) CONGRESS NEWS acquaints readers with the content and conclusions of important congresses and seminars at home and abroad. e) ASSOCIATION NEWS reports on the work of Slovene biology associations. 2. Originality of Articles Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously published material and should not be under consideration for publication elsewhere. 3. Language Articles and notes should be submitted in English, or as an exception in Slovene if the topic is very local. As a rule, congress and association news will appear in Slovene. 4. Titles of Articles Titles (in Slovene and English) must be short, informative, and understandable. The title should be followed by the name and full address of the author (and if possible, fax number and e-mail address). 5. Abstract The abstract must give concise information about the objective, the methods used, the results obtained, and the con- clusions. The suitable length for scientific articles is approximately 250 words, and for brief note articles, 100 words. 6. Keywords There should be no more than ten (10) keywords; they must reflect the field of research covered in the article. Authors must add keywords in English to articles written in Slovene. 7. Introduction The introduction must refer only to topics presented in the article or brief note. 8. Illustrations and Tables Articles should not contain more than ten (10) illustrations (graphs, dendrograms, pictures, photos etc.) and tables, and their positions in the article should be clearly indicated. All illustrative material should be provided as physical originals (photographs or illustrations). Tables with their legends should be submitted on separate pages (only horizontal lines should be used in tables). Titles of tables should appear above the tables, and titles of photographs and illustrations below. Titles of tables and illustrations and their legends should be in both Slovene and English. Tables and illustrations should be cited shortly in the text (Tab. 1 or Tabs. 1–2, Fig. 1 or Figs. 1–2; Tab. 1 and Sl. 1). 9. Conclusions Articles shall end with a summary of the main findings which may be written in point form. 54 Acta Biologica Slovenica, 51 (1), 2008 10. Summary Articles written in Slovene must contain a more extensive English summary. The reverse also applies. 11. Literature References shall be cited in the text. If a reference work by one author is cited, we write allan (1995) or (allan 1995); if a work by two authors is cited, (trinajstić & Franjić 1994); if a work by three or more authors is cited, (pullin & al. 1995); and if the reference appears in several works, (honsig-erlenburg & al. 1992, WarD 1994a, allan 1995, pullin & al. 1995). If several works by the same author published in the same year are cited, the individual works are indicated with the added letters a, b, c, etc.: (WarD 1994a,b). If direct quotations are used, the page numbers should be included: toman (1992: 5) or (toman 1992: 5–6). The bibliography shall be arranged in alphabetical order beginning with the surname of the first author followed by the year of publication, the title of the article, the international abbreviation for the journal (periodical), the vo-lume (in bold print), the number in parenthesis, and the pages. Examples: honsig-erlenburg W., k. krainer, p. milDner & C. Wieser 1992: Zur Flora und Fauna des Webersees. Carinthia II 182/102 (1): 159–173. trinajstić & j. Franjić 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253–256. WarD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10–15. WarD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10–15. Books, chapters from books, reports, and congress anthologies use the following forms: allan J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & Hall, London, 388 pp. pullin A. S., I. F. G. Mclean & M. R. Webb 1995: Ecology and Conservation of Lycaena dispar: British and European Perspectives. In: Pullin A. S. (ed.): Ecology and Conservation of Butterflies, 1st ed. Chapman & Hall, London, pp. 150–164. toman M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s posvetovanja DZVS, Gozd Martuljek, pp. 1–7. 12. Format and Form of Articles Articles should be send as Word document (doc) or Rich text format (rtf) using “Times New Roman CE 12” font with double spacing, align left and margins of 3 cm on A4 pages. Paragraphs should be separated with an empty line. The title and chapters should be written bold in font size 14. All scientific names must be properly italicized. Used nomenclature source should be cited in the Methods section. Tables and illustrations shall accompany the texts separately. All pages including tables and figures should be numbered. The original ma- nuscript, two copies, and an electronic copy (after all corrections) on a 3.5” computer diskette, on CD-ROM or by e-mail must be given to the editor-in-chief. All articles must be proofread for professional and language errors before submission. 13. Peer Review All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene and one foreign) and Brief Note articles by one Slovene expert in the field. Authors may nominate a foreign reviewer in an accompanying letter. Reviewed articles accepted for publication shall be corrected by the author. Authors shall receive thirty (30) free copies of the journal upon publication. In the event an article is rejected, the original material shall be returned to the author together with the negative determination of the editor-in-chief.