ACTA BIOLOGICA SLOVENICA ISSN 1408-3671 UDK 57(497.4) izdajatelj/publisher Dru{tvo biologov Slovenije prej/formerly BIOLOŠKI VESTNIK VOL. 48 ŠT. 1 LJUBLJANA 2005 stevilka 1_05.qxp 13.12.2005 12:32 Page 1 Acta Biologica Slovenica Glasilo Dru{tva biologov Slovenije – Journal of Biological Society of Slovenia Izdaja – Published by Dru{tvo biologov Slovenije – Biological Society of Slovenia Glavni in odgovorni urednik – Editor in Chief Mihael Jo`ef Toman, e-mail: mihael.toman@uni-lj.si Tehni~ni urednik – Managing Editor Branko Vre{, e-mail: branevr@zrc-sazu.si Uredni{ki odbor – Editorial Board Matija Gogala (SI), Nada Gogala (SI) Peter Ma~ek (SI), Alenka Malej (SI), Andrej Martin~i~ (SI), Harald Niklfeld (A), Livio Poldini (I), Boris Sket (SI), Robert Zorec (SI), Mitja Zupan~i~ (SI), Thomas F. J. Martin (USA), Mark Tester (UK), Gerhard Thiel (D) Naslov uredni{tva – Adress of Editorial Office Acta Biologica Slovenica, Ve~na pot 111, SI-1001 Ljubljana, Slovenija http://bijh.zrc-sazu.si/abs/ Oblikovanje – Design @are Vrezec ISSN 1408-3671 UDK 57(497.4) Natisnjeno – Printed on: 2005 Tisk – Print: Tiskarna Ple{ko d.o.o., Ljubljana Naklada: 500 izvodov Cena letnika (dve {tevilki): 3.500 SIT za posameznike, 10.000 SIT za ustanove [tevilka poslovnega ra~una 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 2005 Vol. 48, [t. 1 : 3-48 stevilka 1_05.qxp 13.12.2005 12:32 Page 2 The influence of zinc on the accumulation of cadmium and copper in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda) Vpliv cinka na akumulacijo kadmija in bakra pri kopenskem enakono`cu Porcellio scaber (Crustacea, Isopoda) Primo` ZIDAR Department of Biology, Biotechnical Faculty, University of Ljubljana, Ve~na pot 111, SI-1000 Ljubljana, Slovenia; e-mail: primoz.zidar@bf.uni-lj.si Abstract. Relative to data derived from single-metal exposure not much is known about metal kinetics in terrestrial isopods exposed to a mixture of metals. In the work presented the accumulation pattern of Zn, Cd and Cu were studied in Porcellio scaber, one of the most investigated isopods. Animals were fed with hazel leaves dosed with single metals or their binary mixtures. After twenty-one days of exposure, food consumption and metal accumulation were measured. Results revealed that the accumulation of Zn is not affected by a decreased food consump- tion rate or by possible interactions between the mixture constituents. In contrast, the accumulation of Cd and Cu is significantly reduced when food is also contam- inated with Zn. The lower accumulation of Cd and Cu can be ascribed to interac- tions with Zn that affect uptake and/or loss of metals. Keywords: Isopods, Porcellio scaber, metal mixtures, zinc, copper, cadmium, food consumption, accumulation Izvle~ek. Kinetika kovinskih ionov pri kopenskih rakih enakono`cih, ki so izpostavljeni zmesi kovin je, v nasprotju z izpostavitvijo le eni kovini, malo poz- nana. V predstavljenem delu smo spremljali akumulacijo cinka, kadmija in bakra pri kopenskem raku enakono`cu vrste Porcellio scaber. Poskusne `ivali smo hranili z listi leske, ki smo jim dodali raztopino ene ali pa zmes dveh kovin. Po enaindva- jsetih dneh izpostavitve smo izmerili koli~ine zau`ite hrane in koli~ino akumuli- ranih kovinskih ionov. Rezultati so pokazali, da na akumulacijo cinka ne vpliva upad prehranjevanja kakor tudi ne interakcije med kovinami v zmesi. Nasprotno je akumulacij kadmija in bakra zna~ilno ni`ja ob so~asni prisotnosti cinka v hrani. Ni`ja akumulacija kadmija in bakra je posledica njune interakcije s cinkom, kar vpliva na manj{i privzem ali pa ve~je izlo~anje kadmija in bakra iz telesa. Klju~ne besede: raki enakono`ci, Porcellio scaber, zmesi kovin, cink, baker, kadmij, prehranjevanje, akumulacija kovin ACTA BIOLOGICA SLOVENICA LJUBLJANA 2005 Vol. 48, [t. 1: 3-12 Sprejeto (accepted): 2005-09-27 stevilka 1_05.qxp 13.12.2005 12:32 Page 3 Introduction Terrestrial isopods have been successfully adopted as monitor organisms in assessing the bioavailability of metals (WIESER & AL. 1976, COUGHTREY & AL. 1977, HOPKIN & AL.1986, DALLINGER & AL. 1992, HOPKIN & AL.1993, CORTET & AL. 1999, PAOLETTI & HASSALL 1999). Many investigations have proved that they accumulate the highest tissue concentrations of zinc, copper, lead, and cadmium known for any invertebrate (HOPKIN 1989, CORTET & AL. 1999, HEIKENS & AL. 2001). Their main metal storage organ is the hepatopancreas (WIESER 1961, COUGHTREY & AL. 1980, HOPKIN & MARTIN 1982) where metals are bound to specific low-molecular-weight peptides (DONKER & AL 1990, @NIDAR{I~ 2003), or stored as undissolved metal granules (WIESER & KLIMA 1969, PROSI & DALLINGER 1988, HOPKIN 1989). The hepatopancreas of terrestrial isopods consists of two cell types in which two types of intracellular granules have been observed; B granules in S cells and C granules in B cells (WIESER & KLIMA 1969, HOPKIN & MARTIN 1982, PROSI & DALLINGER 1988, HOPKIN 1989, HOPKIN & AL. 1989). In isopods collected from contaminated sites both types of granules contain Zn whereas only B granules contain Cu and Cd (HOPKIN 1989). Zn stored in B cells can be excreted as these cells show a daily cycle of apocrine secretion whereas Cd and Cu stored in S cells cannot be excreted because S cells have primarily a storage function and were never observed to secrete material into the gland lumen (HOPKIN 1990, HAMES & HOPKIN 1991a). The digestive system is the main route for metal intake in terrestrial isopods. Metal uptake from food depends on the availability of metals (MARTIN & AL. 1976), the number of micro-organisms in the gut and food (COUGHTREY & AL. 1980), the nutritional status (FARCAS & AL. 1996), the rate of food consumption (DROBNE & HOPKIN 1995, BIBI~ & AL. 1997), the temperature (DONKER & AL. 1998), pH inside the gut (HOPKIN 1989), and some other factors. Relative to data derived from single-metal exposures not much is known about the metal uptake in terrestrial isopods exposed to mixtures of metals. Interactions between metals in a mixture may affect the bioavailability of a single metal (VAN GESTEL & HENSBERGEN 1997) and the accumulation and excretion kinetics may differ when animals are exposed to various metals simultaneously (WITZEL 2000, ODENDAAL & REINECKE 2004). As isopods in an industrially polluted environment are usually exposed to several metals at the same time (BEYER & AL. 1984, HOPKIN 1989), some further investigations with metal mixtures are needed. In the present work, we studied the accumulation of Zn, Cd and Cu in one of the most investi- gated isopods, Porcellio scaber, after exposure to single metals or their mixtures. We aimed to show the importance of such investigations for better understanding of metal accumulation and excretion kinetics in isopods and for proper interpretation of biomonitoring data. Materials and methods Experimental animals and procedure Specimens of Porcellio scaber raised in the laboratory were used in the experiments. The parental population was collected in an unpolluted environment in the vicinity of Ljubljana, Slovenia. Juvenile isopods of 15-25 mg fresh weight were placed in plastic Petri dishes (diameter 9 cm), three animals per dish (N=36 for each concentration). A filter paper moistened with water was added to each Petri dish to assure constant humidity. Animals were fed with hazel leaves (Corylus avellana) dosed with a single metal (Zn, Cd, Cu) or binary mixtures (Zn+Cd, or Zn+Cu) for 21 days. The leaves were col- lected from an uncontaminated area in autumn. Solutions of ZnCl2 (>98 % pure Merck, Darmstad, Germany), CuCl2*2H2O (≥99 % pure Merck, Darmstad, Germany) and CdCl2*H2O (>98 % pure Merck, Darmstad, Germany) were applied to the leaves by spraying. The amount of solution applied Acta Biologica Slovenica, 48 (1), 2005 4 stevilka 1_05.qxp 13.12.2005 12:32 Page 4 to each leaf was adjusted to give the following nominal concentrations: Zn – 1000 and 3500 mg kg-1 dry weight; Cu – 500 and 2000 mg kg-1 dry weight; Cd – 250 and 500 mg kg-1 dry weight. Actual con- centrations were analysed and did not differ of those desired by more than 5%. The base concentra- tions of Zn, Cu and Cd in the control leaves from the collection site were 40 mg Zn kg-1 dry weight, 20 mg Cu kg-1 dry weight and 0.15 mg Cd kg-1 dry weight. Control animals were fed with untreated food. The experiment was performed in a climate chamber at a relative humidity of 100% and at 16 hours light and 8 hours dark regime. Temperature was kept constant at 21°C (±1°C). Food consumption was measured as the difference in the weight of leaves at the beginning and at the end of the experiment. Food consumption rates (CR) were calculated as the absolute consump- tion of food per week divided by the dry weight of the animals. Metals concentration analyses After exposure to the metal treated food, animals were food deprived for 24 hours to empty their guts. Then they were lyophilised, weighed and completely digested in a nitric/ perchloric acid mix- ture (7:1). After evaporation of the acid, the residue was taken up in 0.1% HNO3. Total Zn, Cu and Cd concentrations in whole animals were determined by flame atomic absorption spectrometry (Perkin Elmer AAnalyst 100). As certified reference material a Dogfish Liver (DOLT-2, National Research Council Canada) was used. The leaves were analysed for metals in the same way. Statistical analyses of data Data on metal accumulation and food consumption rates were analysed using two-way ANOVA with the factors Zn concentration in food (3 levels), Cd concentration in food (3 levels), Cu concen- tration in food (3 levels), and the food consumption rate as the covariate (the criteria for significance: p<0.05). For each metal concentration or combination used accumulation of a single metal was cor- related with food consumption rate by using Pearson’s correlation coefficient (the criteria for signif- icance: p<0.05). For all statistical analyses the SPSS 12.0 for Windows software was used. Results Accumulation of cadmium and zinc The concentration of Cd in control animals and animals fed with only Zn-dosed food did not exceed 2 mg kg-1 dry body weight. Animals exposed to food contaminated with 250 and 500 mg Cd kg-1 dry food weight accumulated up to 400 mg Cd kg-1 dry body weight (Fig. 1). Cadmium accumu- Primo` Zidar: The influence of zinc on the accumulation... 5 0 50 100 150 200 250 300 350 400 450 500 0 Cd 250 Cd 500 Cd C d (m g/ kg dw ) 0 Zn 1000 Zn 3500 Zn Figure 1: Concentration of Cd in Porcellio scaber after three weeks exposure to food contaminated with Zn or Cd or their mixture (AVR±SE). stevilka 1_05.qxp 13.12.2005 12:32 Page 5 lation from food was significantly lower where Cd in food was combined with Zn (Tab. 1). Cadmium accumulation was in general correlated with the food consumption rate which decreased with the increased concentration of both Cd and Zn in food (Fig. 3). No correlation between accumulated Cd and accumulated Zn was found. Accumulation of Zn (Fig. 2) was not dependent on food consumption rate (Tab. 1 and Fig. 3). In contrast to Cd, the accumulation of Zn from food even slightly increased when offered in a mixture with Cd (Fig. 2). Accumulation of copper and zinc The concentration of Cu in control animals and animals fed with Zn-dosed food was around 200 mg kg-1 dry body weight (Fig. 4). The copper concentration in Cu treated animals increased with increasing Cu concentration in food. Animals exposed to food contaminated with 3500 mg Zn kg-1 food in a mixture with 500 or 2000 mg Cu kg-1 food accumulated significantly less Cu compared to animals fed with only Cu-contaminated food. 1000 mg Zn kg-1 food did not affect the accumulation Acta Biologica Slovenica, 48 (1), 2005 6 Table 1: ANOVA (p values) for Zn, Cd and Cu accumulation in Porcellio scaber after 21 days of exposure to food dosed with a single metal (Zn, Cd, or Cu) or binary metal mixtures (Zn+Cd or Zn+Cu). 0 100 200 300 400 500 600 700 800 0 Zn 1000 Zn 3500 Zn Zn (m g/ kg dw ) 0 Cd 250 Cd 500 Cd Figure 2: Concentration of Zn in Porcellio scaber after three weeks exposure to food contaminated with Zn, Cd, or their mixture (AVR±SE). stevilka 1_05.qxp 13.12.2005 12:32 Page 6 of Cu. In general, the food consumption rate had no significant effect on the accumulation of Cu, although the consumption rate decreased with increasing concentration of Zn and Cu in food (Fig. 6, Tab. 1). On the other hand, in animals exposed to 500 mg Cu kg-1 + 3500 mg Zn kg-1 food, Cu accumulation significantly correlated with food consumption rate. The accumulation of Zn was affected neither by the Cu concentration in food nor by the food consumption rate (Tab. 1) and increased with increasing concentration of Zn in food (Fig. 5). Primo` Zidar: The influence of zinc on the accumulation... 7 0 1 2 3 4 5 6 0 Cd 250 Cd 500 Cd C R (m g/ m g dw ) 0 Zn 1000 Zn 3500 Zn Figure 3: Food consumption rate (CR) in Porcellio scaber after three weeks exposure to food contaminat- ed with Zn, Cd, or their mixture (AVR±SE). 0 200 400 600 800 1000 1200 0 Cu 500 Cu 2000 Cu C u (m g/ kg dw ) 0 Zn 1000 Zn 3500 Zn Figure 4: Concentration of Cu in Porcellio scaber after three weeks exposure to food contaminated with Zn, Cu, or their mixture (AVR±SE). 0 100 200 300 400 500 600 700 800 0 Zn 1000 Zn 3500 Zn Zn (m g/ kg dw ) 0 Cu 500 Cu 2000 Cu Figure 5: Concentration of Zn in Porcellio scaber after three weeks exposure to food contaminated with Zn, Cu, or their mixture (AVR±SE). stevilka 1_05.qxp 13.12.2005 12:32 Page 7 Discussion Our results demonstrated lower accumulation of Cd and Cu in Porcellio scaber after exposure to food dosed with Zn+Cd or Zn+Cu mixtures compared to single metal exposure. In contrast, the accu- mulation of Zn was nearly unaffected by the presence of Cd or Cu. The changed accumulation pat- tern for Cd and Cu can be attributed to a changed food consumption rate or to interactions between the mixture constituents. Metal intake may be avoided by regulation of the food consumption rate (DROBNE & HOPKIN 1995, ZIDAR & AL. 2003b) or by food selection (KASCHL & AL. 2002, ZIDAR & AL. 2003a, 2004, 2005). The two selected concentrations of Zn, Cd, and Cu used in our experiment were previously determined as the concentrations with no major impact on food consumption rate and as concentra- tions that decrease the food consumption rate in short term exposure to a single metal (ZIDAR 2000). In contrast to our previous finding, 1000 mg Zn /kg and 500 mg Cu /kg were recognised as concen- trations that already affect food consumption rate. In a mixture, all metal concentrations decreased food consumption. At all concentrations used the food consumption rate was correlated with accu- mulation of Cd, but not with Cu or Zn accumulation, although the latter had the strongest effect on food consumption rate. This proved that Cd accumulation could be reduced by the regulation of food consumption rate, as found in our previous studies (KASCHL & AL. 2002, ZIDAR & AL. 2003ab, ZIDAR & AL. 2005). Beside food consumption rate, chemical and physiological interactions between food con- stituents might also affect metal bioavailability. It is known that Cd has a higher affinity for chloride ions than Zn (reviewed in RAINBOW 1997) which affects the uptake of Cd from the substrate in collembolans (VAN GESTEL & HENSBERGEN 1997). In isopods Cd complexation with chloride most probably does not affect the uptake of Cd. HAMES & HOPKIN (1991b) found an even higher assimi- lation rate for Cd compared to Zn when both metals were offered in a mixture as chloride salts (100 mg Zn/ kg + 100 mg Cd/ kg dry food weight). In Porcellio laevis exposed to food contaminated with a Zn and Cd sulphate mixture, lower accumulation of Cd was observed (ODENDAAL & REINECKE 2004). Copper is not known to complex with chloride but it has stronger affinity for organic ligands than Zn (HERTZ & AL. 1990). POSTHUMA & AL. (1997) reported that Cu reduced the sorption of Zn to soil whereas Cu sorption was inert toward Zn addition. In contrast to our findings, Cu also stimulates the uptake of Zn in Enchytraeus crypticus (POSTHUMA & AL.1997). Elevated concentrations of Zn might either reduce Cd and Cu uptake or increase their loss from storage sites in P. scaber. The only significant route by which metals can be assimilated or excreted Acta Biologica Slovenica, 48 (1), 2005 8 0 1 2 3 4 5 6 0 Cu 500 Cu 2000 Cu C R (m g/ m g dw ) 0 Zn 1000 Zn 3500 Zn Figure 6: Food consumption rate (CR) in Porcellio scaber after three weeks exposure to food contaminat- ed with Zn, Cu, or their mixture (AVR±SE). stevilka 1_05.qxp 13.12.2005 12:32 Page 8 in terrestrial isopods is via the digestive system: via the cells of the hepatopancreas and papilate region of the gut (HOPKIN & MARTIN 1984, HAMES & HOPKIN 1989). There are two potential major mechanisms for the uptake of dissolved trace metals in crustaceans; binding to metal-specific mem- brane carriers, or entry via routes used in the uptake of major ions like Ca (RAINBOW 1997). The later route is known for Cd (WRIGHT 1977), whereas Zn uptake does not appear to follow any route for major ion uptake (RAINBOW & DALLINGER 1993). Evidence from studies on invertebrates suggests that assimilation of Zn into cells is regulated metabolically whereas Cu transport is strictly passive, dependent solely on the extracellular-intracellular gradient (summarised in HOPKIN 1993). Once in the cell, Cu and Cd are bound to a sulphur-containing ligand whereas Zn is bound to phosphate (HOPKIN 1993). DONKER (1992) found that the more Zn is stored in the hepatopancreas of P. scaber the lower is the proportion of Cd accumulated in this organ, while the concentration of Cd in the remainder of the body increased. DONKER (1992) assumed a limited storage capacity for metals in the hepatopancreas. The study of WITZEL (2000) revealed a significant increase in uptake and excre- tion of Cd and Zn when exposed to Zn and Cd simultaneously. WITZEL (2000) assumed that com- bined contamination with Cd and Zn inhibits the complete translocation of the assimilated metals into the hepatopancreas. Probably a fraction of Zn and/or Cd remains in the haemolymph or in other tissues, which can be excreted more easily (WITZEL 2000). This means that Zn might compete with Cu or Cd for metal-specific membrane carriers or for binding sites in metal binding proteins when entering the cell. Conclusions In Porcellio scaber the accumulation of Zn is not affected by decreased food consumption rate or by possible interactions between mixture constituents. In contrast, the accumulation of Cd and Cu is significantly reduced when food is also contaminated with Zn. The lower accumulation of Cd and Cu can be ascribed to metal interactions that affect uptake and/or loss of metals and which should be taken into account in biomonitoring data evaluation. Acknowledgements This work was financially supported by the Slovenian Ministry of Education, Science and Sport (projects No. Z1-3189 and PO-0525). Povzetek Kopenski raki enakono`ci so pomembni pokazatelji obremenjenosti okolja s kovinami, saj kovine v telesu kopi~ijo. Kovine sprejemajo prete`no s hrano, skozi prebavilo, skladi{~ijo pa zlasti v celicah prebavnih `lez, vezane na nizko molekularne beljakovine ali pa v netopnih granulah. Koli~ina privzetih kovin je odvisna od koli~ine zau`ite hrane, koli~ine mikroorganizmov v hrani in prebavilu, dostopnosti kovin v okolju, pH prebavnih sokov, temperature v okolju in drugih dejavnikov. Interakcije med sestavinami hrane in kovinami lahko vplivajo na dostopnost, akumu- lacijo in izlo~anje kovin. Podatkov o medsebojnem vplivu razli~nih kovin v zmesi na privzem posamezne kovine je malo. Kopenski enakono`ci so v industrijsko onesna`enem okolju obi~ajno izpostavljeni visokim koncentracijam ve~ kovin hkrati, zato so raziskave o medsebojnem vplivu kovin potrebne. V predstavljenem delu smo spremljali akumulacijo cinka, kadmija in bakra pri kopenskem raku Primo` Zidar: The influence of zinc on the accumulation... 9 stevilka 1_05.qxp 13.12.2005 12:32 Page 9 enakono`cu vrste Porcellio scaber po izpostavitvi eni kovini ali pa dvem kovinam hkrati. Tovrstne raziskave so pomembne za razumevanje kinetike kovin pri rakih enakono`cih ter za pravilno vred- notenje okoljskih podatkov. V poskusih smo uporabili mladi~e kopenskih enakono`nih rakov vrste Porcellio scaber, ki so bili vzgojeni v laboratoriju. @ivali smo 21 dni hranili z listi leske (Corylus avellana), ki smo jim dodali vodno raztopino cinka, bakra ali kadmija (Zn – 1000 in 3500 mg kg-1 suhe te`e hrane; Cu – 500 in 2000 mg kg-1 suhe te`e hrane; Cd – 250 in 500 mg kg-1 suhe te`e hrane) ali pa zmesi cinka in kad- mija oziroma cinka in bakra. Primerjali smo koli~ino akumuliranih kovin v odvisnosti od koncen- tracije kovin v hrani in koli~ine zau`ite hrane v ~asu poskusa. Pri `ivalih, ki so bile izpostavljene le eni od kovin je telesna vsebnost kovin nara{~ala s koncen- tracijo kovine v hrani. Z nara{~ajo~o koncentracijo kovin je upadala koli~ina zau`ite hrane, kar je vplivalo na privzem kadmija, na privzem cinka in bakra pa ni imelo zna~ilnega vpliva. Akumulacija bakra in kadmija je bila zna~ilno ni`ja ob prisotnosti cinka v hrani, medtem ko na akumulacijo cinka prisotnost bakra ali kadmija ni vplivala. Rezultati so tako pokazali, da med cinkom in kadmijem ter cinkom in bakrom verjetno prihaja do interakcij, zaradi ~esar se privzem kadmija in bakra zmanj{a. Prav tako interakcije med cinkom in kadmijem ter cinkom in bakrom lahko vplivajo na kinetiko asimiliranega kadmija in bakra zaradi ~esar je izlo~anje prese`nih koncentracij slednjih uspe{nej{e. Omenjene ugotovitve je potrebno upo{tevati pri vrednotenju okoljskih podatkov. Literature: BEYER W.N., G.W. MILLER & E.J. 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Ser., Lewis Publishers, pp. 119-131. 11Primo` Zidar: The influence of zinc on the accumulation... stevilka 1_05.qxp 13.12.2005 12:32 Page 11 RAINBOW P.S. 1997: Ecophysiology of trace metals uptake in Crustaceans. Estuar. Coast. Shelf. S. 44: 169- 175. VAN GESTEL C.A.M. & P.J. HENSBERGEN 1997: Interaction of Cd and Zn toxicity for Folsomia candida Willem (Collembola: Isotomidae) in relation to bioavailability in soil. Envir. Tox. Chem. 16: 1177-1186. WIESER W. & J. KLIMA 1969: Compartmentalization of copper in the hepatopancreas of isopods. Microscopie 24: 1-9. WIESER W. 1961: Copper in isopods. Nature 191:1020. WIESER W., G. BUSCH & L. BÜCHEL 1976: Isopods as indicators of copper content of soil and litter. Oecol. 23: 107-114. WITZEL B: 2000: The influence of zinc on the uptake and loss of cadmium and lead in the woodlouse, Porcellio scaber (Isopoda, Oniscidea). Ecotox. Environ. Safety. 47: 43-53. WRIGHT D.A. 1977: The effect of calcium on cadmium uptake by the shore crab Carcinus maenas. J. Exp. Biol. 67: 163-173. ZIDAR P. 2000: Effects of metal ions on food consumption of terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Ph.D thesis. Ljubljana ZIDAR P., D. DROBNE, J. [TRUS & A. BLEJEC 2003b: Intake and assimilation of Zn, Cu and Cd in the ter- restrial isopod Porcellio scaber Latr. (Crustacea, Isopoda). Bull. Environ. Contam. Toxicol. 70: 1028 – 1035. ZIDAR P., D. DROBNE, J. [TRUS, C.A.M. VAN GESTEL & M. DONKER 2004: Food selection as a means of Cu intake reduction in the terrestrial isopod Porcellio scaber (Crustacea, Isopoda). Appl. Soil Ecol. 25 (3): 257-265. ZIDAR P., I.U. KASCHL, J. [TRUS, D. DROBNE & J. BO`I~ 2003a: Avoidance behaviour in Oniscus asellus (Crustacea, Isopoda) as an indicator of food quality. Arch. Ind. Hyg. Toxicol. 54, 177-181. ZIDAR P., J. BO`I~ & J. [TRUS 2005: Behavioural response in the terrestrial isopod Porcellio scraber (Crustacea) offered a choice of uncontaminated and cadmium-contaminated food. Ecotox. (Lond.) 14: 1-10. @NIDAR{I~ N. 2003: Copper and zinc dynamics and related cellular mechanisms in the digestive system of isopod crustaceans Porcellio scaber (Crustacea: Isopoda). Ph.D thesis, Ljubljana. Acta Biologica Slovenica, 48 (1), 2005 12 stevilka 1_05.qxp 13.12.2005 12:32 Page 12 Analysis of Cell Motility with a Successive Sequence of Images Analiza gibanja celic s serijo zaporednih fotografij Katarina ZLATOLAS, Peter VERANI^*, Kristijan JEZERNIK Institute of cell biology, Lipi~eva 2, Medical Faculty of Ljubljana, Ljubljana * corresponding author Abstract. Analysis of cell motility usually requires several hours of continuous video recording of a single view field. Consequently, the method is very much time consuming and only a small number of cells is usually included into analysis. To increase the number of cells monitored in an experiment we developed a simple and expeditious method for analysing the cell motility. A combination of succes- sive series of images of individual cells relocated on CELLocate coverslips and vector graphic editing software Adobe Illustrator 9.0 enabled simultaneous mon- itoring of locomotion for up to ten times more cells as with previous methods. Keywords: Cell motility, digital camera, successive sequence of images, soft- ware Adobe Illustrator 9.0. Izvle~ek. Analiza celi~nega gibanja ponavadi zahteva ve~urno snemanje enega vidnega polja z video kamero. Metoda je zato zamudna in le malo celic se lahko vklju~i v analizo. Z namenom pove~anja {tevila celic v dolo~enem poizkusu, smo razvili preprosto in hitro metodo za analizo celi~nega gibanja. S kombinacijo zapored- nega slikanja istega vidnega polja, kar nam je omogo~ila uporaba CELLokate krovnih stekelc, in vektorskega grafi~nega programa Adobe Illustrator 9.0, smo lahko v anali- zo gibanja celic vklju~ili do desetkrat ve~ celic, v primerjavi z dosedanjimi metodami. Klju~ne besede: Celi~no gibanje, digitalni fotoaparat, zaporedna serija pos- netkov, Adobe Ilustrator 9.0 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2005 Vol. 48, [t. 1: 13-20 Sprejeto (accepted): 2005-09-27 Introduction Monitoring of cell motility provides information about the level of cell differentiation, its respon- siveness to growth factors and can determine at which phase of cell cycle the cell is (BONNETON & al. 1999). The motility of cells is involved in many physiological (embryonic development) and pathological processes (invading malignant cells, immune response and wound repair). Epithelial cells are very much restricted in their motility, because of their attachment to the neighbouring cells and to the extracellular matrix. In order to move from their original place to the final destination, the stevilka 1_05.qxp 13.12.2005 12:32 Page 13 epithelial cells have to go through epithelio-mesenchymal transformation (EMT). Cells, that under- go EMT, become motile and assume fibroblast appearance. Mesenchymal cells have a specialized ability to move through the extracellular matrix, they have front-to-back end polarity and form only transient contacts with their neighbours and the extracellular matrix (HAY 1995). For the measurement of cell motility, various methods have been used recently, mainly using video microscopy and analysis of a sequence of images taken at specific intervals (MORTON & TCHAO 1994, WICK & al. 2003). The common problem for all these methods is that they require monitoring of the same view field for a prolonged period of time due to slow motility of cells. Recently, cover slips with a microgrid became available. They enable relocation of individual cells and consecutive monitoring of several view fields within the time frame of a single experiment. We have developed a method for measurement of cell motility, where a series of consecutive shots with digital camera can be taken on several view fields within the time frame of a single exper- iment. Series of images, analysed with vector graphic editing software, precisely show the path, direction and speed of individual cell. Materials Cell culture Cultures of mice urothelial cell line (g/G) were maintained in 1 : 1 mixture of cell culture medi- um DMEM and HAM F12 with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenit, 100 ng/ml hydrocortisone in 10 % fetal calf serum. All chemicals were obtained from SIGMA Chemical Co. (St. Louis, MO). Cells were grown on microgrid cover slips (Eppendorf CELLocate Coverlips, square 55 µm) in plastic dishes (radius = 2 cm). Motility was stimulated with FGF1 (20 ng / ml) for 48 hours. Between photographic expositions, the cell cultures were maintained in incubator at 37 °C. The cells were outside the incubator, during taking images, for the maximum of 5 minutes. The room temperature was between 28 °C and 30 °C. CELLocate coverslips CELLocate is a round coverslip made of glass in accordance with ISO 8255. The diameter of the coverslip is 12 mm. CELLocate is suitable for light, fluorescence and electron microscopy. Coverslips are available in two different grid sizes: the 55 µm grid size (measured between the inner edges of the measuring square) is suitable for relocation of individual cells, while the 175 µm grid size is more suitable for relocation of cell groups. In this study, we have used coverslips with 55 µm grid size. The grid is composed of 4 x 6 squares and marked alphanumerically. Acta Biologica Slovenica, 48 (1), 2005 14 Figure 1: A microgrid on a CELLocate coverslip. The arrow indicates a 55 µm distance. Slika 1: Mikromre`a na CELLocate krovnem stekelcu. Pu{~ica ozna~uje 55 µm razdaljo. stevilka 1_05.qxp 13.12.2005 12:32 Page 14 Consecutive photography with digital camera Digital camera NIKON COOLPIX 950 attached to inverted phase contrast microscope (NICON TE 300) was used. A low-density culture of g/G cells grown on CELLocate coverslips, with the measuring grid, was used for the analysis of cell motility. The areas with 3 – 5 cells per square were selected. The same area on CELLocate coverslips was relocated every 30 minutes for 5 hours and 30 minutes and as a result, 12 images of an individual view field were obtained. Results The analysis of a sequence of consecutive images with Adobe Illustrator 9.0 The sequence of images, taken with digital camera, were copied to the hard disk of a personal computer. For the analysis of the motility a vector graphic editing software Adobe Illustrator 9.0 was used. The analysis of cell motility was performed as follows. The working sheet was resized to 55 x 55 mm in order to adjust it to the size of CELLocate microgirds square, which was 55 x 55 µm (Fig. 3) Katarina Zlatolas, Peter Verani~, Kristijan Jezernik: Analysis of Cell Motility with a Successive Sequence... 15 Figure 2: A cell grown on a microgrid of CELLocate coverslip. An arrow points to the nucleus. Slika 2: Celica na mre`i CELLocate krovnega stekelca. Pu{~ica ka`e jedro celice. Figure 3: Resizing the working sheet: File > New > Document Set-up > Units = mm > Width = 55 mm > Height = 55 mm. Slika 3: Umerjanje velikosti lista: Datoteka > Nova > Nastavitev dokumenta > Enote = mm> Širina = 55 mm > Vi{ina = 55 mm stevilka 1_05.qxp 13.12.2005 12:32 Page 15 During the next step, a sequence of images were added using the »new layer« for each photo- graph in the sequence and borders of the microgrid square were aligned to the borders of the work- ing sheet (Figs. 4 and 5). A contour of a nucleus of individual cell was made on each picture by using graphic tools (Fig.6). Each picture in a sequence was labelled and listed (Fig. 7 and 8). Acta Biologica Slovenica, 48 (1), 2005 16 Figure 4 and 5: Serial addition of the pictures: Window > Show Layers > Layer 1 > File > Place (place 1st picture ) (Fig. 4) > New Layer > Layer 2 (place second picture) (Fig 5). Sliki 4 in 5: Zaporedno dodajanje slik: Okno> Poka`i plasti > Plast 1 > Datoteka > Postavi (postavimo prvo sliko) ( Slika 5) > Nova plast > Plast 2 (postavimo drugo sliko) (Slika 5). stevilka 1_05.qxp 13.12.2005 12:32 Page 16 The use of different colours (Window > Show Colour), transparency (Window > Show Transparency) and/or darker outlines (Window > Show Colour) is recommended to distinguishing partly overlapping nucleus positions in slow moving cells. The procedure was repeated for all twelve images taken. Each photograph was deleted after con- touring the nucleus and only contours were retained on the layers to reduce the size of the file (Fig. 9). In the end, all the layers were made visible on the monitor and the path of cells was analysed. Katarina Zlatolas, Peter Verani~, Kristijan Jezernik: Analysis of Cell Motility with a Successive Sequence... 17 Figure 6: Borders adjustment: Window > Show Tools > Selection Tool (an arrow) > press and hold »shift« button and draw the corner of the picture with an arrow to adjust the borders of microgrid and working sheet. Slika 6: Prilagoditev robov: Okno > Orodja > Izbrano orodje (pu{~ica) > ob so~asnem dr`anju tipke »shift« s pu{~ico vle~emo vogal fotografije, da se pokrijeta robova delovnega lista in kvadrata mikromre`e Figures 7 and 8: A contour of a nucleus: Tool > Ellipse Tool > (Fig.7) draw a contour of a nucleus > click in the centre of the ellipse > (Fig. 8) choose the width and length of the ellipse (2 x 2 mm) > Window > Show Colour > Selection Tool > choose the new ellipse (circle) > colour the circle > Type Tool > write the serial number of the picture Sliki 7 in 8: Obrisovanje jedra: Orodja > Elipsa > (Slika 7) nari{i obris jedra > klikni v sredino elipse > (Slika 8) izberi {irino in vi{ino elipse (2 x 2 mm) > Okno > Poka`i barve > Izbrano orodje > izberemo novo elipso > obarvamo elipso > ^rkovno orodje > vpi{emo zaporedno {tevilko slike stevilka 1_05.qxp 13.12.2005 12:32 Page 17 While the distance between the two points, showing position of the nucleus in 30 minutes intervals, was short (7-9 µm), the path between the points was estimated to be straight. The velocity of an individual cell was calculated from the distance the cells reached in a definite time interval. The path was manually measured on a printed cumulative image, containing the posi- tions of the nuclei at 30 minutes intervals. The system error was calibrated by 12 measurements of the distance between the same two points (from the centre to the centre of two points). The error in measurements of the path between the two points was ± 0.3 µm. Discussion The aim of the present study was to find an exact and quick method for the measurement of indi- vidual cell migration. The system of consecutive images and vector graphic editing software Adobe Illustrator 9.0 was used. The problem in analysing epithelial cell migration is that an average speed of motility is approx- imately 20 µm per hour, so at least 5 hours of monitoring is required for the analysis of the motility of cell moving in a single view area (BRAY 1992). Thus, in most studies the number of monitored cells was limited less than eighth cells within an experiment (MORTON & TCHAO 1994, WICK & al. 2003). With our method, using series of successive images of cells on defined areas of a cover- slip with a micro-grid, motility of more than 80 cells (20 cells per experimental group) was analysed within the time frame of a single experiment. The increased number of monitored cells makes the results of measurements more accurate. In our experiments, where the velocity of urothelial cell line g/G was measured after stimulation with FGF1, we used 30 minutes long intervals, because we ascertained that this is the optimal inter- val for the velocities close to 20 µm / h. In 30 minutes, the centre of gravity of the fastest cells Acta Biologica Slovenica, 48 (1), 2005 18 Figure 9: Result of 12 consecutive positions of a cell nucleus. Bar = 10µm Slika 9: Prikazanih je 12 zaporednih polo`ajev jedra gibajo~e se celice. Merilna ~rtica = 10 µm stevilka 1_05.qxp 13.12.2005 12:32 Page 18 changed their position for 10 µm, which is approximately half of the cell length. Therefore, the 30 minutes long interval between snap-shots is long enough for monitoring of five view fields with min- imal error. Because of various velocities of cell motilities in different cell types and frequent changes of direction, shorter intervals are appropriate for the analysis of higher velocity. However, shorter intervals reduce the number of experimental groups, which can be monitored simultaneously. During motility, cells undergo dramatic phenotype changes that are mainly based on the dynam- ic assembly and disassembly of actin filaments underlying the plasma membrane (BRAY 1992). Cells that become motile usually assume a fibroblastic appearance with lamellipodia adjusting the shape by each change of the motility direction (BONNETON & al. 1999, BRAY 1992). Such changes of the cell shape make motility of different parts of the cell inhomogeneous and so difficult to analyse. The problem is usually solved by determining nucleus as the centre of gravity. Because this most prominent organelle of eukaryotic cells, lies in the area, where usually most of the cell mass is concentrated (BONNETON & al. 1999). In our experiments, a contour of nucleus was drawn with Adobe Illustrator 9.0 software. The nuclei of our cells are usually round, which simplifies drawing a contour. The monitoring of an individual cell nucleus displacement was enabled by the use of CELLocate coverslips with a microgrid. Until now, the displacement of each individual cell has usually been followed using recording tape of time-lapse video recorder, tracing paper and curvimeter. Due to better resolution compared to most video cameras, which were mainly used in such studies (BONNETON & al. 1999, MOR- TON & TCHAO 1994, WICK & al. 2003), makes digital camera better choice for the analysis of cell motility. Better resolution is important for the accurate localization of the nuclei taken as the centre of cell gravity. During cell division, the nuclear envelopes become fragmented and reorganization of cytoskele- ton appears. Cells become rounded, reduce the attachment surface and become immobile until the end of mitosis (BONNETON & al. 1999). Occasionally such dividing cells detach from the surface and become lost. Changing of cell morphology and motility during cell division represents an obsta- cle in measurements of velocity. For this reason, it is of benefit to monitor as many cells as possible. Certain cell lines do not adhere well to glass and therefore, do not grow well on CELLocate. In such cases, Eppendorf Company recommends that CELLocate is coated with collagen. Using colla- gen is not appropriate for the coverslips with 55 µm grid size, because it reduces the clarity and vis- ibility of cell nucleus. The margins of 175 µm grid size coverslips are deeper, which enhance the con- trast and so enable the use of collagen coating. This method for analysing the cell motility with a combination of successive series of images of individual cells and vector graphic editing software Adobe Illustrator 9.0 has revealed itself as very successful, while being quick and exact. Most of all it enables simultaneous monitoring of locomo- tion for much larger number of cells in comparison with previously used methods. Povzetek V dosedanjih raziskavah so za spremljanje gibanja celic uporabljali predvsem videomikroskopi- jo. Vse dosedanje metode omogo~ajo spremljanje gibanja celic le v enem vidnem polju za vsak eksperiment. Zaradi majhne hitrosti gibanja celic je spremljanje celic zamuden proces, zato je ve~ina analiz omejena na majhno {tevilo celic, kar zmanj{a verodostojnost dobljenih rezultatov. Z namenom, da bi lahko hkrati spremljali gibanje celic na ve~ vidnih poljih, smo razvili metodo Katarina Zlatolas, Peter Verani~, Kristijan Jezernik: Analysis of Cell Motility with a Successive Sequence... 19 stevilka 1_05.qxp 13.12.2005 12:32 Page 19 zaporednega fotografiranja z digitalnim fotoaparatom in analizo hitrosti gibanja z grafi~nim vek- torskim programom Adobe Illustrator 9.0. Med celi~nim gibanjem se oblika celice spreminja, zato smo spremljali spremembe polo`aja te`i{~a izbranih celic. Za te`i{~e celice smo dolo~ili vedno bolj ali manj okroglo jedro, ki ponavadi le`i v podro~ju, kjer je zbrana glavnina mase celice. Namesto videokamere smo uporabili digitalni fotoaparat, ki zaradi bolj{e lo~ljivosti omogo~a natan~nej{e meritve. V dolo~enih ~asovnih zamikih smo izbrana polja s celicami zaporedoma fotografirali in tako dobili serijo slik, ki ponazarjajo smer in hitrost gibanja dolo~ene celice. Ponovna lokalizacija celic in merjenje premika celice je omogo~ila uporaba CELLocate krovnih stekelc z jed- kano mre`o. Serija dobljenih posnetkov individualnih celic predstavlja prepotovano pot celice, iz katere lahko izra~unamo hitrost potovanja. Predstavljena metoda omogo~a spremljanje celic na ve~ vidnih poljih isto~asno, kar lahko bistveno pove~a {tevilo opazovanih celic in s tem relevantnost rezultatov merjenja hitrosti gibanja celic. References BONNETON C., J.B. SIBARITA & J.P. THIERY 1999: Relationship between cell migration and cell cycle dur- ing the initiation of epithelial to fibroblastoid transition. Cell Motility and the Cytoskeleton 43: 288-295. BRAY D. 1992: Cell movements. Garland publishing, INC., New York & London, pp. 17-45. HAY E.D. 1995: An overview of epithelio-mesenchymal transformation. Acta Anat. 154: 8-20. MORTON D.M. & R. TCHAO 1994: Regulation of motility and cytoskeletal organization of rat bladder car- cinoma cells by cyclic AMP. Cell Motility and the Cytoskeleton 29: 375-382. WICK N., S. THURNER, K. PAIHA, R. SEDIVY, I. VIETOR & L.A. HUBER 2003: Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis. Springer- Verlag: Histochemistry and Cell Biology, pp. 1-12. Acta Biologica Slovenica, 48 (1), 2005 20 stevilka 1_05.qxp 13.12.2005 12:32 Page 20 The macrophytes of lake Velenjsko Jezero, Slovenia – the succession of macrophytes after restoration of the lake Makrofiti Velenjskega jezera, Slovenija – sukcesija makrofitov po restavraciji jezera Zdenka MAZEJ1, Mateja EP[EK2 1 ERICo Velenje; Environmental Research and Industrial Co-operation Institute, Koro{ka 58, 3320 Velenje, Slovenia; Tel.: +386 3 898 19 58; fax: +386 3 898 19 42; E-mail: zdenka.mazej@erico.si 2 Biotechnical Faculty, Department of Biology, Ve~na pot 111, 1000 Ljubljana, Slovenia Abstract. The macrophyte vegetation in the artificial lake Velenjsko jezero has been monitored since 1996. The pH of the lake was around 12 up to 1994, when it was remediated. After that macrophytes started to colonize a large proportion of the littoral very quickly. The pioneer species, which appeared in the first survey year (1996), were Chara sp., Nuphar lutea, Potamogeton crispus and Myriophyllum spi- catum. Potamogeton crispus was the dominant species till 1997. In the following years Najas marina and Potamogeton filiformis prevailed over other species in the lake. Since the beginning of the colonization, the species composition had become more heterogeneous and the quantitative relationships between the species varied enormously. In the year 2004, 9 species were detected in the lake. While species Najas marina, Potamogeton filiformis, Potamogeton lucens, Potamogeton nodosus show positive development progress, in the last years minor appearance of Chara sp., Potamogeton crispus and Najas minor was observed. Key words: lake Velenjsko jezero, succession of macrophytes, species compo- sition and distribution, relative plant mass, maximal depth colonization Izvle~ek. Makrofite v umetno nastalem Velenjskem jezeru spremljamo od leta 1996. Vrednost pH jezerske vode je bila vse do leta 1994, ko so v Termoelektrarni [o{tanj uvedli zaprti krog transportne vode, okoli 12. Vrednost pH je po tem ukre- pu hitro padla na vrednost okoli 8. Makrofiti so postopoma pri~eli naseljevati litoralno obmo~je jezera. Pionirske vrste v letu 1996 so bile Chara sp., Nuphar lutea, in Potamogeton crispus. Potamogeton crispus je bila dominantna vrsta vse do leta 1997. V naslednjih letih sta prevladali vrsti Najas marina in Potamogeton filiformis. Od za~etka naseljevanja je prihajalo do velikih kvalitativnih in kvantita- tivnih sprememb v vrstni sestavi in razporeditvi makrofitov. V jezeru je leta 2004 ACTA BIOLOGICA SLOVENICA LJUBLJANA 2005 Vol. 48, [t. 1: 21-31 Sprejeto (accepted): 2005-09-27 stevilka 1_05.qxp 13.12.2005 12:32 Page 21 uspevalo 9 vrst. Medtem ko ka`ejo vrste Najas marina, Potamogeton filiformis, Potamogeton lucens in Potamogeton nodosus pozitiven trend v razvoju, pa v zadnjih letih opa`amo manj{e pojavljanje vrst Chara sp., Potamogeton crispus in Najas minor. Key words: Velenjsko jezero, sukcesija makrofitov, vrstna sestava, distribucija makrofitov, rela- tivna rastlinska biomasa, maksimalna globina naselitve Introduction The succession of submersed macrophytes in lakes is a complex process. Once they are estab- lished, macrophytes have significant feedback effects on lake water quality and ecosystem health, and are an important consideration in lake management (JEPPESEN & al. 1998). In lakes where con- ditions favour development of high biomass of submerged aquatic vegetation over large areas, there is potential for large-scale reductions in water column concentrations of nutrients and phytoplank- ton, and increase transparency (JEPPESEN & al. 1998). Factors that influence the biomass and distri- bution of submersed macrophytes both among and within lakes have been well studied. These fac- tors include substratum character and water depth (WEISNER 1991), transparency (HUTCHINSON 1975, NICHOLS 1992, MIDDELBOE & MARKAGER 1997, MAZEJ & GABER{~IK 1999, VAN DUIN & al. 2001, BEST & al. 2001), water chemistry (PIP 1984, HASLAM 1987, PRESTON 1995, GERM & GABER{~IK 1996, GERM & al. 2003), temperature (PIP 1989, ROONEY & KALFF 2000), bottom slope (NICHOLS 1992), water level fluctuation and wave action (PRESTON 1995, ANDERSSON 2001), filamentous and planktonic algae (URBANC-BER~I~ & al. 2002), grazing (JACOBSEN & SAND-JENSEN 1992, WEISNER & al. 1997) and intra- and inter- species competition among macrophyte species (FOX 1992, AGAMI & WAISEL 2002). The underwater light regime is one of the most important determinants of submerged aquatic vegetation (HUTCHINSON 1975, NICHOLS 1992, BEST & al. 2001, VAN DUIN & al. 2001). DUARTE AND KALFF (1986) asserted that within lakes, the morphometric variables of bottom slope and community exposure are both negatively correlated with macrophyte biomass. In lakes, which shelve rapidly, the density of plants on the shore of the lake falls very quickly. On the contrary, if the shore is shallow and slopes gently, the belt overgrown by aquatic plants can be very wide (NICHOLS 1992). Many macrophytes are tolerant to pH values between 10 and 11; higher values are destructive (BOWES 1987). The pH of Lake Velenjsko jezero was around 12 until 1994, when reconstruction of the fly ash system and introduction of a closed loop water cycle was made at the [o{tanj Thermal Power Plant. The pH declined to around 8 and biota started to colonize the lake. Our study was focused on aquatic macrophytes, an important group of primary producers, which provide conditions necessary for fish, zooplankton and benthos in the littoral zones of lakes. Information on the distri- bution and species composition of aquatic vegetation is necessary for management and understand- ing of aquatic systems. We monitored the succession of species composition and abundance of sub- mersed macrophytes after restoration measures in Lake Velenjsko jezero. Our method was based on direct field observations along transects that enabled identification of the main environmental factors influencing the appearance of macrophytes. Acta Biologica Slovenica, 48 (1), 2005 22 stevilka 1_05.qxp 13.12.2005 12:32 Page 22 Materials and Methods Description of the site Velenjsko jezero is located in the [alek Valley, at an altitude of 366 m, with a surface 135000 m2 and a maximal depth of 54 m. It is an artificial lake resulting from mining activity. As a result of sub- sidence, whole settlements, meadows and fields submerged and flooded. Up to 1983 fly ash slurry from the [o{tanj Thermal Power Plant was transported by pipeline and emptied into Velenjsko jeze- ro. It brought ash and calcium hydroxide to the lake, raising the pH of lake water to 12. Since 1983 the ash was used to build in embankments, but effluent with a pH around 12 remained the predom- inant polluter of the lake until 1994. After reconstruction of the fly ash system and a closed loop water cycle (October 1994), biota appeared in the lake again. It was recolonized by phyto- and zoo- plankton, fish, macrophytes and other organisms. The pH of the lake is now around 8. The lake shore is sparsely overgrown with emergent plants (e.g. Typha latifolia L., Phragmites australis (Cav.) Trin. Ex Steud., Schoenoplectus lacustris (L.) Palla [Scirpus lacustris L.]); the major part of the lake shore consists of bare areas, meadows and allotments. The littoral zone of the lake can be divided into three main regions: the fairly steep western and south-western unstable shoreline, a north-eastern marshy area, and the eastern and southern part of the lakeshore, which is used for sunbathing, walking, rid- ing, bicycling and other recreational activities. Near the shore are allotments. Macrophyte cover The method of surveying the entire littoral from a boat, using depthmeter, view box and sam- pling rake, was applied in the years 1996, 1997, 1999, 2000, 2003 and 2004. In the year 2003 sur- veying of macrophyte community was carried out in detail. The littoral was checked every month from June to September. The shoreline was divided into 25 sections of the length 200±2 m. Species abundance in each section was evaluated according to KOHLER & JANAUER (1995) on a five level descriptor scale (1 – very rare, 2 – infrequent, 3 – common, 4 – frequent, 5 – abundant, predomi- nant). The evaluated quantity of plants is interpreted as mass index (MI), which is with »real bio- mass« (PM) related with the function PM = MI3. Obtained data has been processed by standard methodology, made in co-operation between the University teams of Hohenheim (Germany) and Vienna /Austria) (KOHLEER AND JANAUER 1995). The Internet based description of the methodology is accessible on the internet side www.midcc.at. From the data we can calculated two mass indexes; MMO is the Mean Mass Index of the individual species with respect to the survey transects they occur, MMT is the Mean Mass Index of individual species with regard to the full length of the lake shoreline. The distribution ratio »d« of each species stands for MMT3/MMO3. Nearly similar and high values of MMT and MMO (»d« value is near 1) reveal that a species is abundant and that it occurred in almost all sections – the distribution of the species is homogeneous. Zdenka Mazej, Mateja Ep{ek: The macrophytes of lake Velenjsko jezero... 23 MIi = Mass Index of species in transect i Ali = length of transect i, where species occurred GL = total length of lake shoreline stevilka 1_05.qxp 13.12.2005 12:32 Page 23 The Relative Plant Mass (RPM %) was used to calculate the quantitative significance of indivi- dual species in a section (PALL & JANAUER 1995). PMxi = MIxi 3 RPMx = relative plant mass of species x MIxi = for any transect i estimated mass of the species x PMxi = plant mass of species x in transect i Li = length of lake transect i j = running index of the different species Physical analyses of water: Water transparency was measured with a Secchi disk. Temperature at 30 cm and pH were meas- ured with a MultiLine P4. Results After restoration of the lake Velenjsko jezero, macrophytes, almost instantaneously, started to gradually overgrow large surface of the littoral. In the Tab. 1, presence and relative abundance of dif- ferent macrophyte species in lake Velenjsko jezero for the period 1996 – 2004 is presented. The pio- neer species that appeared in the year 1996 were Chara sp., Nuphar lutea (L.) Sibth. & Sm. and Potamogeton crispus L.. They colonized only a minor part of the littoral. Only three years later almost the whole littoral was overgrown and the species composition had become more heterogeneous. Changes in quantitative relationships between the species varied as well. Potamogeton crispus was the dominant species till 1997. In the following years Najas marina All. and Potamogeton filiformis Pers became the most abundant species in the lake. Acta Biologica Slovenica, 48 (1), 2005 24 Species / Year of survey Abbrev. 1996* 1997 1999 2003 2004 Jun Jul Sept Aug Jun Jul Aug Jun Jul Aug Chara sp. Cha sp 1 - - - - - - 1 1 1 Myriophyllum spicatum L. Myr spi - 2 2 2 2 2 1 2 2 2 2 Najas marina All. Naj mar - - - 3 4 1 3 4 - 2 4 Najas minor All. Naj min - - - 2 3 - - - - 1 2 Nuphar lutea (L.) Sibth. Et Sm. Nup lut 1 1 1 1 1 1 1 1 1 1 Potamogeton crispus L. Pot cri 4 3 1 1 2 1 1 3 1 1 Potamogeton filiformis Pers. Pot fil - 1 - - 1 2 3 3 3 3 3 Potamogeton lucens L. Pot luc - - - - 2 1 2 2 1 1 2 Potamogeton nodosus Poir. Pot nod - - - - 2 1 1 1 1 1 1 * Relative abundance of the species was not defined. Table 1: Presence and relative abundance of different macrophyte species in lake Velenjsko jezero for the period 1996 – 2004. Presence: ♦ – present, – – not present. Relative abundance: a five level descriptor scale (1 – very rare, 2 – infrequent, 3 – common, 4 – frequent, 5 – abundant, predominant) (KOHLER & JANAUER 1995). Tabela 1: Prisotnost in relativna abundanca posameznih makrofitskih vrst v Velenjskem jezeru v letih med 1996 in 2004. Prisotnost: ♦ – prisotna – – ni prisotna. Relativna abundanca vrst: petstopenjska lestvica (1 = zelo redka; 2 = redka; 3 =zmerno zastopana; 4 = pogosta; 5 = prevladujo~a vrsta) (KOHLER & JANAUER 1995). stevilka 1_05.qxp 13.12.2005 12:32 Page 24 From the Fig. 1, where the distribution and relative abundance of different macrophyte species around lake Velenjsko jezero is presented for August 2003, it is evident that Najas marina and Potamogeton filiformis were the most abundant species in the lake in the late summer. It can be seen from Fig. 2 that the quantitative relation among species during the growth season varied enormous. Species Potamogeton filiformis was dominant in June, while Najas marina was the predominant species in the lake in the late summer (RPM was 68.5% in August and 84.6% in September). Other species were much less abundant. Zdenka Mazej, Mateja Ep{ek: The macrophytes of lake Velenjsko jezero... 25 Figure 1. Distribution and abundance of different macrophyte species around lake Velenjsko jezero in August 2003. The Lake was divided into 25 section with the length of 200±2 m. Slika 1: Distribucija in abundanca razli~nih makrofitskih vrst v Velenjskem jezeru avgusta 2003. Jezero smo razdelili na 25 transektov dol`ine 200±2 m. Figure 2. Relative plant mass (RPM %) throughout the season 2003 - Quantitative relation among species during the growth season. Slika 2: Spreminjanje razmerja v relativni rastlinski biomasi (RPM %) med razli~nimi vrstami makrofi- tov od junija do septembra 2003. stevilka 1_05.qxp 13.12.2005 12:32 Page 25 From the high »d« values, near 1 (Fig. 3) it is evident that Najas marina and Potamogeton fili- formis overgrew most of the littoral in August and September, but the share of Potamogeton filiformis was much smaller than that of Najas marina. The RPM of Potamogeton filiformis was only 25.2 % in August and 8.2 % in September (Fig. 2). Tab. 2 presents some physical characteristics of the water of lake Velenjsko jezero, in the years 1997, 2000, 2003 and 2004. The mean pH of the lake water was around 8 without any significant variations. The mean Secchi disk transparency (Zs) was the highest in 1997, but in the next years it decreased, while the mean temperature of the water increased. Most macrophyte species colonized only shallow part of the littoral in 1997 (Tab. 3). This year, species Potamogeton crispus reached the maximum depth of 3.5 m. Four species (Najas marina – Zc = 4.0 m, Myriophyllum spicatum – Zc = 4.0 m, Potamogeton lucens – Zc = 4.0 m and Acta Biologica Slovenica, 48 (1), 2005 26 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 Myr spi Pot fil Pot luc Pot nod Pot cri Naj mar Nup lut Macrophyte species "d " va lu e Jun Jul Aug Sept Figure 3. The distribution ratio - »d« values (the ratio between MMT3 and MMO3) for different species throughout the season 2003. Slika 3: Distribucijski kvocient - »d« vrednost (razmerje med MMT3 in MMO3) za razli~ne makrofitske vrste preko sezone 2003. Year Sechi disc transparency (m) Temperature (oC) pH P-tot. (mg/l) N-tot. (mg/l) 1997 5.9 (4.2/9.0) 19.8 (17.8/21.3) 8.3 (8.2/8.7) 0.24 (0.13/0.44) 2.0 (1.47/2.57) 2000 4.5 (3.2/5.0) 22.2 (19.8/25.4) 8.4 (8.2/8.8) - - 2003 4.6 (4.0/5.5) 23.4 (19.4/25.8). 7.8 (7.4/8.3) 0.1 (0.1/0.1) 1.3 (0.9/2.1) 2004 3.7 (3.0/4.5) 21.7 (19.0/23.7) 8.2 (8.1/8.2) 0.1 (0.1/0.1) - Table 2: Mean physical and mean chemical characteristic of water in lake Velenjsko jezero averaged over the period May to September in the years 1997, 2000 and 2003; average value (minimum value/maxi- mum value). Tabela 2: Povpre~ni rezultati fizikalnih meritev in kemijskih analiz vode v Velenjskem jezeru v ~asu od maja do septembra v letih 1997, 2000, 2003 in 2004; povpre~je (minimalna/maksimalna vrednost). stevilka 1_05.qxp 13.12.2005 12:32 Page 26 Potamogeton filiformis – Zc = 5.5 m) colonized the deepest part of the littoral in the year 2003. From the data of the Table 3, negative relationship between Zs and maximum colonization depth (Zc) was calculated – correlation coefficient Zs/Zc: r = – 0.59, n= 14. Discussion Lake Velenjsko jezero could be classified as meso-eutrophic according to the level of total phos- phorus (OECD 1982, MAZEJ & GABER{~IK 1999), what offered good conditions for quick coloniza- tion of macrophytes after restoration of the lake. Lake Velenjsko jezero, because of its original basin shape, has a steep slope in its littoral along the majority of the lakeshore. This is the reason why only a narrow area of shoreline is colonized by submersed macrophytes. ANDERSSON (2001) asserted that the geomorphology of a lake basin is the primary factor affecting the structure of the interface zone between land and water. The steepness of the lake margins is decisive for sediment stability along slope gradients, which in turn influences the establishment of vegetation. The pioneer species, which colonized only a minor part of the littoral in the year 1996, were Chara sp., Nuphar lutea and Potamogeton crispus (MAZEJ 1998). Only three years later these species are not important compo- nents of the littoral vegetation anymore. Chara sp. disappeared from the lake when other macrophyte species overgrew the littoral in 1999, but it was detected in a very small quantity again in 2004. It is known that Charophytes are pioneers among macrophytes, and colonize a new habitat very quickly (PALMA-SILVA & al. 2002), but they are usually not competitive with angiosperms (BLINDOW 1992). Nuphar lutea has remained more or less at the same abundance at the same location throughout these years. Floating-leaved species may be expected to have a faster occupancy of areas with shallower water and smaller fetches, and the development of the floating-leaved macrophyte community may require years (REA & al. 1998). It has been reported (NICHOLS & SHOW 1986, BOLDUAN & al. 1994, JIAN & al. 2003) that Potamogeton crispus and Myriophyllum spicatum are very invasive species, which usually form a very dense population and establish large monospecific weed beds in many lakes. However, this was not the case in lake Velenjsko jezero. Potamogeton crispus was the domi- nant species till 1997. The life history of Potamogeton crispus differs from most other submersed Zdenka Mazej, Mateja Ep{ek: The macrophytes of lake Velenjsko jezero... 27 Zs = 5.9 m Zs = 4.6 m Chara sp. 0.5 - Myriophyllum spicatum L. 3.0 4.0 Najas marina All. 2.0 4.0 Najas minor All. 1.5 - Nuphar lutea (L.) Sibth. Et Sm. 2.0 2.0 Potamogeton crispus L. 3.5 3.0 Potamogeton filiformis Pers. 0.3 5.5 Potamogeton lucens L. - 4.0 Potamogeton nodosus Poir. - 2.0 Table 3: Maximum colonization depth (Zc) of seven macrophyte species in 1997 and in 2003; Zs (mean Secchi depth). Tabela 3: Najve~ja globina uspevanja (Zc) sedmih makrofitskih vrst v letu 1997 in v letu 2003; Zs (povpre~na Secchi-jeva globina). stevilka 1_05.qxp 13.12.2005 12:32 Page 27 plants. Biomass production of Potamogeton crispus often reaches its maximum in early summer, allowing it to avoid competition from other species in the habitat primarily because they are still in the dormant state (TOBIESSEN & SNOW 1984). Young plants of Potamogeton crispus and Myriophyllum spicatum overwinter, and then grow rapidly in the spring. In this respect, the two species are similar (BOLDUAN & al. 1994). By early summer plants undergo senescence processes and then remain dormant until autumn. It is known that Potamogeton crispus grows well in eutroph- ic lakes, at low temperature and very low light intensities, being an important primary producer in freshwater ecosystems, providing a good food source for herbivorous fishes (JIAN & al. 2003). Grazing by fish, birds and invertebrates may be an important factor that limits development of plants. Grazing sensitivity differs among submersed macrophytes (JACOBSEN & SAND-JENSEN 1992, WEISNER & al. 1997), and it has been proposed that selective grazing affects species composition and even succession (CRAWLEY 1983). It was ascertained that species of genus Potamogeton were signif- icantly more heavily grazed by invertebrates (mean 4.2 % of leaf area) than non-Potamogeton species (mean 0.8 % of leaf area) (JACOBSEN & SAND-JENSEN 1992). The grazing experiments made by Weisner & al. (1997) also showed that non-Potamogeton species like Chara sp. and Myriophyllum spicatum were not significantly affected by grazing. There are no data in the literature about her- bivory on the species Najas marina, but we assume that this species is not tasteful food for herbi- vores due to its fragility and its prickly shoots. Fragile shoots are easily broken and spread around by the help of current, wind and birds. It might be the reason, that Najas marina predominates over other species in lake Velenjsko jezero, including Myriophyllum spicatum. Myriophyllum spicatum and Najas marina usually appear together in various natural habitats, where they compete with each other. The result of studies (AGAMI & WAISEL 2002) showed that Myriophyllum spicatum was more sensitive to intraspecific competition than Najas marina, and additionally the competitive effects of Najas marina on Myriophyllum spicatum were stronger than vice versa. Light availability affects both the biomass and community structure of phytoplankton and sub- mersed vegetation (VAN DUIN & al. 2001, BEST & al. 2001). Although a significant positive correla- tion between Secchi disc transparency and the maximum colonization depth of macrophytes has been found in several studies (HUTCHINSON 1975, CHAMBERS & KALFF 1985, NICHOLS 1992, MIDDELBOE & MARKAGER 1997, MAZEJ & GABER{~IK 1999), our study showed that transparency was not the main factor which determined the depth distribution of macrophytes in lake Velenjsko jeze- ro. The maximum colonisation depth of macrophytes is usually ascribed to light attenuation in the water column and the minimum light requirement for growth (BLINDOW 1992), although other parameters such as surface irradiance, hydrostatic pressure, water colour, temperature, grazing pres- sure, substrate type and epiphyte loading can also affect maximum colonization depth (SAND-JENSEN 1989, VAN DUIN & al. 2001). Tall macrophytes (caulescent angiosperms and charophytes) compen- sate for light limitation by shoot growth towards the water surface, so maximum colonization depth is therefore independent of transparency (MIDDELBOE & MARKAGER 1997). Although mean Secchi disc transparency was higher in 1997 than in 2003, the maximum colonization depth of macrophytes (Potamogeton crispus) was only 3.5 m in 1997 and 5.5 m in 2003 (Potamogeton filiformis). The results of ROONEY AND KALFF (2000) showed that angiosperms colonized deeper parts during years characterized by an early warming of the water, independent of underwater irradiance. The year 2003 was very warm with high May air temperatures. The average temperature of lake water was 3.6 o C higher than that in 1997. Since the majority of plants reproduce asexually, an earlier start to the grow- ing season allowed plant communities more time for colonization. Acta Biologica Slovenica, 48 (1), 2005 28 stevilka 1_05.qxp 13.12.2005 12:32 Page 28 Conclusion The lake Velenjsko jezero has offered good conditions for development of macrophytes in less than ten years. In this short period, species composition and the quantitative relationships between the species varied enormously. Therefore further changes are expected in the coming years. We assume that plant interactions might constitute the main controlling factor regulating the distribution of macrophytes and coming projects should be oriented in studying plant interaction in grater detail. Povzetek Do leta 1983 je bilo Velenjsko jezero odlagali{~e pepela iz termoelektrarne [o{tanj. Od leta 1983 naprej so s sedimentacijo lo~evali pepel od transportne vode. Pepel so odlagali na deponijo, vodo pa {e naprej ~rpali v jezero, kjer se je pH dvignil na okoli 12 in iz jezera so izginile vse oblike `ivljen- ja. @e eno leto po uvedbi zaprtega kroga transportne vode leta 1994, kjer vodo ~rpajo nazaj in jo ponovno uporabijo, se je pH na povr{ini jezera zmanj{al na 9, leta 1997 pa pH vode po celotnem glo- binskem profilu ni presegal 8,7 in vanj se je po dalj{em obdobju spet za~elo vra~ati `ivljenje. Vrstno sestavo in razporeditev makrofitov v Velenjskem jezeru spremljamo bolj ali manj redno `e od leta 1996. Makrofiti so pri~eli po sanacijskih ukrepih zelo hitro naseljevati prazen litoral, saj so bile nudene ugodne razmere – dosti hranil, podlaga za ukoreninjenje in ugodne svetlobne razme- re. Velenjsko jezero je nastalo z ugrezanjem, zaradi ~esar se njegova obala na mnogih mestih zelo hitro spusti v globino. To je tudi razlog, zakaj je z makrofiti pora{~en le ozek pas litorala. Pionirske vrste, ki so se pojavile v prvem letu raziskav (1996) so bile Chara sp., Nuphar lutea in Potamogeton crispus. Potamogeton crispus je bila dominantna vrsta do leta 1997. V naslednjih letih pa sta vrsti Najas marina in Potamogeton filiformis prevladali nad ostalimi vrstami v jezeru. Chara sp. je bila prisotna v zelo majhni koli~ini v jezeru le v letih 1996 in 1997, v naslednjih letih pa je v ob~asnih pregledih litorala nismo ve~ zasledili. Zopet pa smo jo na{li na istem mestu v letu 2004. V vseh teh letih je prihajalo do spremenjenega kvantitativnega in kvalitativnega razmerja med vrstami. V prvih letih so makrofiti naselili predvsem del med ~olnarno in pritokom Sopote, medtem ko je bil del ob deponiji nepora{~en. V naslednjih letih so se makrofiti raz{irili tudi v ostala obmo~ja litorala, zmanj{ala pa se je njihova pojavnost tam, kjer se ka`e ve~ji vpliv zaledja – ob nasipu in na obmo~ju pe{poti pod vrti~karskim naseljem »Kinte-Kunte«. Od leta 1996 pa do leta 2004 je {tevilo vrst iz 3 naraslo na 9. Medtem ko ka`ejo vrste Najas marina, Potamogeton filiformis, Potamogeton lucens in Potamogeton nodosus pozitiven trend v razvoju, pa v zadnjih letih opa`amo manj{e pojavljanje vrst Chara sp., Myriophyllum spicatum, Potamogeton crispus in Najas minor. Le nekaj let je minilo od naselitve prvih makrofitov v jezeru, zato lahko pri~akujemo, da bo pri{lo v naslednjih letih {e do dolo~enih sprememb in sicer tako v vrstni sestavi makrofitov kot tudi v razporeditvi le-teh. Aknowledgements Authors thank to Prof. Dr. Alenka Gaber{~ik and Dr. Mateja Germ for valuable comments on the manuscript. Zdenka Mazej, Mateja Ep{ek: The macrophytes of lake Velenjsko jezero... 29 stevilka 1_05.qxp 13.12.2005 12:32 Page 29 References AGAMI M. & Y. WAISEL 2002: Competitive relationships between two water plant species: Najas marina L. and Myriophyllum spicatum L.. Hydrobiologia 482: 197-200. 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Oecologia 109: 592-599. Zdenka Mazej, Mateja Ep{ek: The macrophytes of lake Velenjsko jezero... 31 stevilka 1_05.qxp 13.12.2005 12:32 Page 31 stevilka 1_05.qxp 13.12.2005 12:32 Page 32 Secular trend in body dimensions in boys from Tuzla region in period 1980 –1996 1HAD@IHALILOVI] Jasminka, 1TERZI] Rifet, 1AHMI] Adisa, 2H.HALILOVI] Amir, 3NO@INOVI] Amra, 1[EHI] Amela, 1HAMIDOVI] Hajrija, and 4JUSUPOVI] Fatima 1Faculty of Natural Science and Mathematics, Department of Biology, University of Tuzla, 75000 Tuzla, Univerzitetska br.4, Bosnia and Herzegovina; Tel.fax: ++ 387 35 286 454; E-mail: jasnahba@yahoo.com 2Gymnasium »M. Selimovi}« Tuzla, Bosnia and Herzegovina; 3School of Phylosophy of Tuzla University, Faculty of Physical Education, Bosnia and Herzegovina; 4College of Healt Studies, University of Sarajevo, Bosnia and Herzegovina. Abstract. Within 16 year-period secular trend in seven measurements of physical growth of male children and youth from Tuzla Region, was researched by correspon- ding analysis of the sample that involved 1329 researched subjects. Our data were compared with the results of research from 1980 in the sample of 1349 boys. Nine suc- cessive generations in 1980 and 1996, from Tuzla region, were involved in this research. Analysis of the data obtained is based primarily on scientific elaboration of the situation registered in 1996 in the tested part of the broader population, after an unnatural and extremely unfavorable period for physical growth of the large majority of that population. The aim of this research was to establish secular trend (negative or positive) for the seven measurements of physical growth of male children and youth comparing our results from 1996 to corresponding research results on growth and development from 1980, the sample of about same population (nearly same number of investigated persons). Although the unfavorable war living conditions negatively affected ontogenesis of the researched subjects, body dimensions of male children and youth were established as harmonious, in the limits of average European standards. However, it seems the unfavorable living conditions caused temporary slowdown in body dimensions, so, for these generations (11 to 19 years old), we could not find any increase of mean values (for certain number) of the researched parameters in compar- ison with the sample from 1980 year. So, 16 year-acceleration trend for most param- eters was established and it is particularly evident in postpubertal period. Key words: secular trend, boys’ growth and development ACTA BIOLOGICA SLOVENICA LJUBLJANA 2005 Vol. 48, [t. 1: 33-44 Sprejeto (accepted): 2004-11-17 Introduction In the last century, nearly in all the world population, gradual acceleration of different increased measures of physical growth was noticed. This occurance was marked as acceleration of growth (sec- stevilka 1_05.qxp 13.12.2005 12:32 Page 33 ular trend), and it means faster growth (shortened growth cycle) faster achievement of sexual matu- rity and increase in body height and weight. Children are »higher« and »heavier« in all ages than the children of the same age 100, 50, even 20 years ago. The term acceleration in broader sense means increase in body dimensions, prolonged reproductive period, longer human lifetime, faster growth of fetus, changes in psychic development, then increased dimensions of certain organs, as well as mat- uration of their functions (NIKOLI} & DOVAT 1978 ). Most significant factors causing this occurance are: improvement of general living conditions (among them nutrition is most important), children get sick more rarely, increased possibility of new combination of genes is present due to more frequent migrations (NIKOLI} & DOVAT 1978), occur- ance of increased number of heterozygotes for the genes that prevailing to a certain extent direct growing. There is no direct evidence that it is a causal mechanism, so environmental factors might be main cause of secular changes (SUSANNE 1985, MALINA 1979). However, secular change of growth is a reversible biological process, so, if socio-economic and growth-hygienic conditions would aggravate to the level they were on a few centuries ago, we could expect come-back of growth type that used to exist in Europe (WIERINGEN 1979). The same author concludes that secular changes may be positive and negative. He relied upon the time of investiga- tion and upon geographic varieties among population (WIERINGEN 1986). This author researched sec- ular changes in Holland, related to the body height, within the period 1850 -1978, and found (1979) strong connection between the secular changes and general socio-economic impacts, nutrition, health conditions, and demographic parametrs. In his opinion there are no indications of certain changes (as a result of selection) in genotype of the Dutch population that could be the cause of that connection. Heterosis did not play a significant role in it, either. For Belgian population the data for the period 1830-1980 show the average height and weight sig- nificantly increased, particularly in the course of the growing period, and to a lower extent, in the adulthood. These data are in accordance to European and North American data on secular trend for the period 1880 –1980, and they are as follows: 1,5 cm/decade in childhood, 2,5 cm/decade in ado- lescence, and about 1 cm/decade in adulthood. Increase in body dimensions of adults in the recent decades has varied between 0,3 & 3,0 cm/decade (HAUSPIE & AL 1997 ). Secular tendency toward a higher growth and earlier maturity are predominantly connected to better nutrition and general health conditions of the population (HISAFUMI & AL 1999). In the last two or three decades in our country 18-years old boys are every year approximatelly high- er for about 0,2 mm, heavier to 0,3 kg (BERBEROVI} & HAD`ISELIMOVI} 1982). This increase is expected to be followed by improvement of socio-economic conditions and vice versa, and it decreases during crises and wartime periods. Some indications show that this rate has decreased since 1980, or even stopped in these countries (BIELICKI & WALISKO 1991; SANNA, FLORIS & COSSEDU 1993). These indications in industrialized countries show that environmental living con- ditions enabled reaching their maximal genetic potentials, or their social conditions ceased improv- ing (WEBER & AL 1995). Secular tendency in the achieved growth and growth rate are usually more frequent in children of lower socio-economic status. Secular trend in growth rate slowdowned in some populations, but in the others it is going on, or it may decrease in future. Lots of researches in the world show that secular tendency in adults’ height has been getting slow in most countries during recent 10 to 20 years (HAUSPIE & AL 1996). The lowest increase in growth of the adults was noticed in Sweden and Norwey, what indicates that adults’ growth nearly reached its peak in these countries in recent decades. Western countries like Belgium, England, The Netherlands, show secular tendency between the values of 1,0 – 1,3 cm/decade. The highest increase in the adults’ height (3,0 cm/decade was found (in women) in Chech Republic and (in men) in Japan (1950 – 1960; HAUSPIE & AL 1996). Many countries worldwide have publications with referential data on children and youth’s body development, and on secular trend in certain populational measures. In former Yugoslavia secular trend in growth and development was researched by KOVA~ (1973), BRODAR (1961), DOVE~AR Acta Biologica Slovenica, 48 (1), 2005 34 stevilka 1_05.qxp 13.12.2005 12:32 Page 34 (1978), TOMAZO-RAVNIK (1988, 1999), [TEFAN~I} ET AL. (1996), GAVRILOVI} (1972, 1974), PREBEG (1978, 1995, 1997), and IVANOVI} (1985). The tables and centile diagrams (obtained in such researches) are used directly as tables of norms in pediatrics, school medicine, in anthropologic sport institutes (because they deal with normal physi- cal growth of children and youth). Basic biological factors of secular tendencies are not completely cleared up. However, most of researchers dealing with this phenomenon have been explaining it by genetic factors and factors of external environment. These data on acceleration of individual physical growth seriously put the question: Will the period required for reaching the boys & girls’ sexual maturity keep shortening childhood and will a human being be of a giantic stature in future? However, if the suppositions on heterosis (as a result of increased migrations) and improved socio-economic situation in the world population are the main factors of this acceleration, then it is difficult to believe in such prognoses. In addition to this, pos- sibility of increasing heterezygosity in stimulation of quantitative features (potentially similar geno- types) is naturally limited, the topic that has been discussed about much recently, (HAD`ISELIMOVI} 1988). HIERNAUX (1975) set a hypothesis that our descendants would probably be higher than we are today, but they should not be giants. Basic demographic, economic and climate features of the researched Region Tuzla is a powerful economic, cultural, educational and administrative center, not only in the Region itself, but on all B&H territory. This city is characterized by relatively fast socio-economic develop- ment after World War II, what resulted in a hasty increase of inhabitants number, primarily by means of mechanical increase in population (migrations). So, the census from 1971 year established the number of 107 293 inhabitants in Tuzla, out of which 37,08% were immigrants. The biggest increase in inhabitants number was registered between 1955 and 1965. This factor influenced bioanthropo- logic heterogeneity of the researched sample of the Tuzla’s population, as well. From the time of the researches in Tuzla community (NOVAKOVI} 1980) and the researches of ours, number of inhabitants increased for 47 092 more. For the time of 16 years (including the war results, as well), demographic picture of Tuzla changed considerably.In the course of the four year- war (1992-1996), the number of inhabitants in this region increased rapidly due to forced migrations, banishments, so, in 1996 it amounted 154 384; 38.566 (24,98% of expatriates, and 115 818 (75,03%) domiciles (FEDERAL BUREAU OF STATISTICS SARAJEVO 1998). Mixture of the population of different local origin with domiciles before the war, as well as expatriates presence and displaced people during and after the war in the Region of Tuzla affected (and will do it in the future) the changes of genetic structure of the population. According to the Federal Bureau of Statistics Sarajevo (1998) there were 31 097 (20,14%) employed people. And 16 years ago the number of employed people was 34 885 (32,50%) in 1980 (NOVAKOVI} 1980). According to the same source, an average salary for December 1996 was 253,10 KM or 126,55 Eura. From the data offered it can be concluded that Tuzla’s populaton is mainly consisted of workers, what naturally affects socio-economic status of a family and the population in general. The war and migrations changed the national structure of the researched Region. Speaking about climate, Tuzla has moderate-continental climate. In this Region, summers are warm, winters are cold. The mean year air temperature is relatively high, it ranges from 10,2 to 11,4 0C, with clearly distinguished seasons. Summer and spring temperatures are changeable (KULENOVI} 1980). Had`ihalilovi} J., Terzi} R., Ahmi} A., H. Halilovi} A., No`inovi} A., [ehi} A.: Secular trend... 35 stevilka 1_05.qxp 13.12.2005 12:32 Page 35 Objectives To analyze 16 year-secular trend in growth dynamic changes by comparison the obtained results with corresponding data from 1980. To create necessary data – base for research of temporal differences, including four-year aggression on Bosnia and Herzegovina, taking into consideration the fact that all the researched subjects lived one period of physical growth in extremely unfavorable war conditions. Material and methods Researches that are a source of data collected in this study, were carried out in Tuzla Region in autumn 1996/97. Anthropological measurements were carried out out in four primary and two sec- ondary schools. Crosss – sectional method was used. The measurement involved 1321 male pupils, age 11 to 19 years.The sample was randomly chosen and stratified according to subjects’ decimal age: pupils age 10 years and six months to the 11 years and 5 months made the group of the age 11 years; 16,6 to 12,5 made the group of the age 12 years (Table 1). Seven anthropometric parameters were used in this research: 1. stature 2. body weight 3. mean chest circumference 4. upper arm circumference; 5. total arm length; 6. biacromial breadth and 7. biil- iocristal breadth. All measurements were carried out according to IBP (International Biologic Program), (HAD`ISE- LIMOVI} & LELO 1998). Descriptive statistics and t-tests were done in the Program Statistics for Windows, Copyright Stat Soft, Inc.1993. Statistical analysis primarily involved evaluation of the central tendency measure- ments. Statistical significance of differences between the results of our research and the researches from 1980 was evaluted, as well (t-test). Results and discussion Stature (body height) is one of the most stable parameters of physical growth, that reflects complex processes in organism. Comparison of the results from 1996 (series II) with those from 1980 (series I) reveals significantly lower mean values of body height in series II than in series I (age 11 to 15 years).This difference in mean values is statistically significant, that is shown in t-test results (Table 2). Acta Biologica Slovenica, 48 (1), 2005 36 1980 1996 1996 Total: Age (years) N N N Domiciles Refugees 11 113 107 32 139 12 149 117 26 143 13 143 132 33 165 14 188 146 38 184 15 140 115 41 156 16 187 75 58 133 17 138 89 57 146 18 192 86 69 155 19 69 54 46 100 Total: 1349 921 400 1321 Table 1: Age structure of the researched sample stevilka 1_05.qxp 13.12.2005 12:32 Page 36 Boys from series II over 15 year age have higher mean body height than in series I, due to 16 year – accelerating trend. So, for example, 18 years old boy from series II is approximately higher for 1,16 cm than the same boy from series I, so, acceleration trend for that generation is 0,73 cm/decade. Secular trend for the generations from 11 to 15 years, is negative (Fig.1) (due to already mentioned reasons), but for the postadolescent growth (age 16, 17, 18 and 19 years) is evident and amounts 1,01 cm/decade. These data correspond to decade diapason of secular trend for Europe and North America, that show secular trend in growth between 1880 and 1980 was 1,5 cm/decade in childhood, 2,5 cm/decade in adolescency, and about 1,0 cm/decade in adulthood (MALINA 1990). This temporary slowdown in body height increase in boys from series II in prepuberty and puberty is probably due to higher child’s sensitivity to negative impact of external factors. Namely, all the external factors that more or less affect physical growth and development, were led to the values that strongly affected children and youth in the mentioned period due to four year-agression. Results on body height in Ljubljana’s boys (Slovenia) from 1991/92, series III ([TEFAN~I} & AL 1996), and from 2000 year – series IV (STREL & AL 2001) show secular trend in that area to stagnate (Fig.1). Figure 1 shows boys from series III and IV approximately »higher« than boys from series II. Those differences in average body height are supposed to be result of genetic potential difference. Had`ihalilovi} J., Terzi} R., Ahmi} A., H. Halilovi} A., No`inovi} A., [ehi} A.: Secular trend... 37 Age N1 1980. N2 1996. X1 (cm) X2 (cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 145,36 142,77 5,98 6,54 - - 3,28* * 12 149 143 150,07 146,30 7,06 6,55 3,24 2,47 4,74* ** 13 143 165 156,02 153,47 8,36 8,40 3,96 4,90 2,66* 14 188 184 163,05 161,13 9,20 8,29 4,50 4,99 2,12* 15 170 156 169,98 168,18 8,03 8,56 4,25 4,37 1,95 16 187 133 170,90 172,44 7,61 6,56 0,54 2,53 1,94 17 138 146 174,07 175,14 6,55 6,99 1,85 1,56 1,33 18 192 155 176,76 177,92 7,16 7,21 1,54 1,59 1,50 19 69 100 176,17 178,87 6,78 6,79 0,99 0,53 2,54* Table 2: Stature of the boys of Tuzla Region (1980 & 1996) 140 144 148 152 156 160 164 168 172 176 180 11 12 13 14 15 16 17 18 19 Age St at ur e (c m ) Series I Tuzla 1980 Series II Tuzla 1996 Series III Ljubljana 1991/92 Series IV Ljubljana 2000 Fig.1. Comparative diagram of stature of Tuzla's male children and youth: 1980 and 1996, and the youth from Ljubljana 1991/92 and 2000 stevilka 1_05.qxp 13.12.2005 12:32 Page 37 Those differences might have been a bit smaller if the boys from series II had not stagnated in growth due to already mentioned reasons. Examples of negative and stagnating secular trend were found in many countries in a lot of studies including those from wartime and economic crises (TANNER 1968, WALKER & AL 1988, KUH AND WADSWORTH 1989). Body weight. In addition to body height, body weight is most important parameter in bioanthropol- ogy for research and monitoring physical growth and development. Unlike height, weight is labile factor, changable by impact of various endo- and exogenous factors. Body weight increase coincides with body hight increase, so most intensive increase in body weight in series II was registered in age of 14 years (Fig. 2). Negative or stagnating secular trend was registered in series II in age of 11, 12, 13, 17, and 18 years in comparison to series I. Comparison of results from series II and series I shows boys from series II, aged 11 to13 years have significantly lower average body weight, than in series II (Table 3, Fig. 2). Temporary stagnation in body weight increase in series II could be explained before all, by incorrect diet (hunger, lack of vitamins and proteins), bad socio-healthy and hygienic conditions our domicile and banished population experinced in the course of the last agression on Bosnia and Herzegovina. Comparison of the results from series II on average body weight and series III and IV (considering time distance between publishd reseraches in Ljubljana and Tuzla) shows differences that are an out- come of already mentioned differences in average body height (Table 3, Fig. 2). Acta Biologica Slovenica, 48 (1), 2005 38 30 35 40 45 50 55 60 65 70 75 11 12 13 14 15 16 17 18 19 Age Bo dy W ei gh t ( kg ) Series I 1980 Tuzla Series II 1996 Tuzla Series III 1991/92 Ljubljana Series IV 2000 Ljubljana Fig.2. Comparative diagram of body weight of Tuzla's male children and youth: 1980 and 1996, and the youth from Ljubljana 1991/92 and 2000 Age N1 1980. N2 1996. X1 X2 SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 35,92 33,92 6,09 6,07 - - 2,60* 12 149 143 40,02 36,44 8,38 6,57 11,41 7,43 4,07*** 13 143 165 44,13 42,28 9,80 10,76 10,26 16,02 1,58 14 188 184 50,98 50,53 0,09 12,30 15,52 19,53 0,5 15 170 156 56,40 55,87 9,46 11,77 10,63 10,56 0,45 16 187 133 58,05 59,98 9,86 9,08 2,92 7,35 1,81 17 138 146 63,50 61,96 8,21 8,79 9,38 3,31 1,53 18 192 155 67,00 66,34 8,72 8,78 5,51 7,06 0,70 19 69 100 68,42 68,35 7,54 9,52 2,11 3,03 0,05 Table 3. Body weight of the boys of Tuzla Region (1980 & 1996) stevilka 1_05.qxp 13.12.2005 12:32 Page 38 Mean chest circumference. Mean chest circumference represents a few parameters of body dimen- sions characteristic for growth. Chest circumference is sufficient if it amounts a half of body height, and insufficient if it is under a half of that measure. Comparison of the results from series II with series I, shows acceleration increase of this parameter (1,78 cm/dec) particularly apparent in post-14 years of life (Table 4). Boys from series II have in all age categories higher average values for mean chest circumference than their age boys 16 years ago (series I). At age 15 to 19 years, the established differences between average values are statistically significant (Table 4). Boys from series II have lower value of chest circumference than in series III in all periods of growth (Fig. 3). Upper arm circumference. Increase in upper arm circumference in series II is highest in adolescent shock (age 14 years), but it is most intensive between age 17 and 18 years, as well, (Table 5). The first maximum in relation to intensive general growth and development in puberty, the second one is likely in relation to muscle growth, when maximal sport and physical efforts start. Secular trend in boys from series II is found in puberty, while it stagnates in prepuberty and postpuberty (Fig. 4). Total arm length. Results of analysis on the arm length are shown in Tabe 6 and Figure 5. Mean values for the arm length are significantly lower (Tab. 6) in series II in relation to series of all ages, except in 18 years old boys. Negative secular trend is happening here until 19 years age, when mean value in series II came close to values in series I. Biacromial breadth. Is a good indicator for growth of trunk, skeleton and musculature. Fig.6 shows that shoulder growth in width is happening in the course of puberty. Most intensive growth is in 15 year age, then it slowdowns until 19 year age. The boys from series II of all ages have significantly higher values (Tab. 7) in relation to series I. Tuzla’s boys (series II) have around same shoulder width as well as their age boys from Ljubljana (series III) in all ages from 11 to 15 years, and after that period Tuzla’s boys have higher values of shoulder width than their age boys from Ljubljana (Fig. 6). Acceleration increase for this parameter is overt in all age categories and it is 1,74 cm/decade. Had`ihalilovi} J., Terzi} R., Ahmi} A., H. Halilovi} A., No`inovi} A., [ehi} A.: Secular trend... 39 Age N1 1980. N2 1996. X1(cm) X2(cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 67,37 68,66 4,15 4,81 - - 2,29* 12 149 143 69,64 70,11 5,12 5,05 3,37 2,10 0,80 13 143 165 72,45 73,80 6,19 6,72 4,04 5,27 1,83 14 188 184 78,74 79,14 4,53 8,22 8,68 7,24 0,58 15 170 156 79,70 82,48 5,42 7,98 1,22 4,21 3,65** 16 187 133 79,76 85,79 4,72 5,65 0,08 4,02 10,07*** 17 138 146 84,11 86,97 4,57 5,77 5,45 1,37 4,64*** 18 192 155 86,03 89,74 4,58 5,29 2,28 3,19 6,90*** 19 69 100 87,49 91,05 4,02 5,48 1,70 1,46 4,86*** Table 4. Mean chest circumference of the boys of Tuzla Region (1980 & 1996) Age N1 1980. N2 1996. X1(cm) X2(cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 19,23 19,41 1,96 1,99 - - 0,72 12 149 143 20,07 19,80 2,42 2,02 2,02 4,36 1,04 13 143 165 20,48 20,76 2,36 2,85 4,84 2,04 0,94 14 188 184 21,81 22,39 2,39 2,98 7,86 6,49 2,08* 15 170 156 22,80 23,26 2,21 2,70 3,87 4,53 1,67 16 187 133 23,30 24,27 2,31 2,25 4,32 2,19 3,77** 17 138 146 24,74 24,37 2,11 2,15 0,41 6,18 1,46 18 192 155 25,29 25,39 2,04 2,13 4,18 2,22 0,44 19 69 100 25,92 25,78 2,01 2,24 1,56 2,49 0,43 Table 5. Upper arm circumference of the boys of Tuzla Region (1980 & 1996) stevilka 1_05.qxp 13.12.2005 12:32 Page 39 Acta Biologica Slovenica, 48 (1), 2005 40 19 20 21 22 23 24 25 26 27 11 12 13 14 15 16 17 18 19 Age U pp er a rm c irc um fe re nc e (c m ) Series I 1980 Tuzla Series II 1996 Tuzla Fig.4. Comparative diagram of upper arm circumference of Tuzla's male children and youth: 1980 and 1996 62 64 66 68 70 72 74 76 78 80 82 11 12 13 14 15 16 17 18 19 Age To ta l a rm le ng th (c m ) Series I 1980 Tuzla Series II 1996 Tuzla Fig.5. Comparative diagram of total arm length of Tuzla's male children and youth: 1980 and 1996 Fig.3. Comparative diagram of mean chest circumference of Tuzla's male children and youth: 1980 and 1996, and the youth from Ljubljana 1991/92 stevilka 1_05.qxp 13.12.2005 12:32 Page 40 Had`ihalilovi} J., Terzi} R., Ahmi} A., H. Halilovi} A., No`inovi} A., [ehi} A.: Secular trend... 41 Age N1 1980. N2 1996. X1(cm) X2(cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 64,04 62,93 3,06 3,52 - - 2,68* 12 149 143 66,25 64,02 3,64 3,29 3,45 1,73 5,49*** 13 143 165 69,52 68,13 4,31 4,15 4,93 6,42 2,87* 14 188 184 72,65 70,98 4,88 4,14 4,50 4,19 3,56** 15 170 156 76,15 74,51 4,32 4,23 4,81 4,97 3,46** 16 187 133 77,14 76,08 3,95 3,63 1,30 2,11 2,48* 17 138 146 78,92 77,21 3,65 3,60 2,30 1,48 3,98** 18 192 155 79,79 78,08 3,59 4,17 1,10 1,13 4,04*** 19 69 100 79,25 78,81 3,66 3,74 0,70 0,93 0,76 Table 6. Total arm length of the boys of Tuzla Region (1980 & 1996) 29 30 31 32 33 34 35 36 37 38 39 40 41 42 11 12 13 14 15 16 17 18 19 Age Bi ac ro m ia l b re ad th (c m ) Series I 1980 Tuzla Series II 1996 Tuzla Series III 1991/92 Ljubljana Fig.6. Comparative diagram of biacromial breadth of Tuzla's male children and youth: 1980 and 1996, with the youth from Ljubljana 1991/92 29 23 22 20 21 24 25 26 27 28 33 32 30 31 34 35 36 37 38 Age Bi iai cr oim l abd oe t lh b(r ) S seoiembI 3879bTuza seoiembI 3885bTuza seoiembIIIb3883/82 Ljudaj n Fig.7. Comparative diagram of biiliocristal breadth of Tuzla's male children and youth: 1980 and 1996, with the youth from Ljubljana 1991/92 stevilka 1_05.qxp 13.12.2005 12:32 Page 41 Biiliocristal breadth. Yearly relative increase in average width of pelvis is highest between 13 and 14 years age (series II; Tab. 8). Pelvis growth completes in 18 years age. Fig.7 shows presence of growth acceleration for this parameter (after 16 years age) in all ages (1,86 cm/decade). Mean val- ues for the pelvis width are significantly higher in series II in relation to series I (confirmed by t-test; Tab. 8). Boys from series II have significantly narrower pelvis than boys from series III in all ages, except for 18 years age, when those values are about equal in both series. Bad living conditions due to the last war did not effect much the width parameters, as they effected body height and circumferences. Conclusions Comparison of our results (series II) and corresponding results (from 16 years ago – series I) shows that boys from series II in prepuberty and puberty for some parameters have same or even lower mean values in relation to the boys from series I (negative acceleration trend). This a direct result of negative effect of exogenous factors that caused temporary growth stagnation in all age groups. In postpuberty that stagnation is relatively fast compensated, so a mild secular trend is present for all parameters observed for that growth and developmental period. All the boys from series II spent one or two intensive periods of heght growth in prewar, wartime and postwar period, so malnutrition, comprehensive indigence and other war misfortunes significantly slowdowned growth and development in both periods.That’s why the most intensive growth was pro- longed for one year more, as well, in relation to the expected one. Acta Biologica Slovenica, 48 (1), 2005 42 Age N1 1980. N2 1996. X1(cm) X2(cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 20,65 21,56 1,54 1,91 - - 4,19*** 12 149 143 21,55 22,20 1,63 1,97 4,35 2,96 3,07** 13 143 165 22,74 23,44 1,87 1,74 5,52 5,59 3,42** 14 188 184 23,82 24,90 2,09 2,03 4,74 6,22 5,09*** 15 170 156 25,19 26,01 2,01 2,01 5,75 4,45 3,66** 16 187 133 25,76 26,82 1,79 1,74 2,26 3,14 5,30*** 17 138 146 26,71 27,48 1,71 1,65 3,68 2,44 3,85** 18 192 155 27,45 27,90 1,74 1,61 2,77 1,52 2,47* 19 69 100 27,53 28,04 1,56 1,64 0,29 0,52 2,05* Table 8. Biiliocristal breadth of the boys of Tuzla Region (1980 & 1996) Age N1 1980. N2 1996. X1(cm) X2(cm) SD1 SD2 Relative Increase (%) 1 2 t-test p<0,05 11 113 139 30,31 31,70 1,72 1,78 - - 9,17*** 12 149 143 31,57 32,30 1,68 1,82 4,15 1,60 3,56** 13 143 165 32,82 33,60 2,43 2,29 3,95 4,02 2,89* 14 188 184 34,39 35,32 2,40 2,66 4,78 4,11 3,54** 15 170 156 36,24 37,42 2,54 2,98 5,37 5,95 3,83** 16 187 133 36,97 38,69 2,40 2,28 2,01 3,38 6,52*** 17 138 146 38,68 39,67 2,15 2,42 4,49 2,52 3,67** 18 192 155 39,41 40,19 2,19 2,21 2,01 1,31 3,29** 19 69 100 40,05 41,03 1,97 2,19 1,62 2,11 3,04** Table 7. Biacromial breadth of the boys of Tuzla Region (1980 & 1996) stevilka 1_05.qxp 13.12.2005 12:32 Page 42 Ten year-secular trend for body height for the boys from series III and IV (Ljubljana) was slow- downed, while 16 year trend for this parameter (series I and II) is evident in postpuberty. Comparison of our results and those of [TEFAN~I} & AL (1996) and of STREL & AL (2001) revealed larger mean values for almost all tested parameters in Ljubljana’s male youth of all tested genera- tions. However, those differences slowly decrease in postadolescent period. We assume those differ- ences occured due to different ecologic factors, living standard, as well as specific genetic features charasteristic for all populations. We suppose the population of the researched area has not come close to full expression of genetic material for the investiagted features. Sa`etak [esnaestogodi{nji trend sedam pokazatelja fizi~kog rasta i razvoja mu{ke djece i omladine prou~avan je odgovaraju}om analizom uzorka, koji je 1996. godine obuhvatio 1329 ispitanika. Na{i podaci kom- parirani su odgovaraju}im rezultatima istra`ivanja iz 1980. godine u uzorku od 1349 dje~aka. U ovu studiju uklju~eno je 9 sukcesivnih generacija iz 1980. i 1996. godine sa podru~ja Tuzle. Provedena anl- iza prikupljenih podataka primarno po~iva na nau~noj elaboraciji registriranog stanja iz 1996.godine u prou~avanom dijelu {ire populacije, nakon jednog neprirodnog i ekstremno nepovoljnog perioda u procesu rasta i razvoja ogromne ve}ine njihovih pripadnika. Cilj ovog rada je bio utvrditi sekularni trend (negativan ili pozitivan) za 7 antropometrijskih pokazatelja rasta i razvoja mu{ke djece i omla- dine, kompariraju}i na{e rezultate iz 1996. godine sa odgovaraju}im podacima rasta i razvoja na uzorku pribli`no iste veli~ine (iste populacije) iz 1980. godine. Unato~ tome {to su nepovoljni (ratni) `ivotni uvjeti negativno djelovali na ontogenezu ispitanika, utvr|eno je da se rast i razvoj mu{ke djece i omladine na prou~avanom podru~ju odvija skladno te da se nalazi u granicama prosje~nih evropskih standarda. Ipak, nepovoljni `ivotni uvjeti izazvali su privremeni zastoj u rastu i razvoju, pa se u pogo|enim uzrasnim kategorijama (od 11 do 15 godina `ivota) nije moglo uo~iti pove}anje srednjih vrijednosti (za jedan broj) izu~avanih parametara u pore|enju sa uzorkom iz 1980. godine. [esnaesto- godi{nji akceleracijski trend za ve}inu parametara je utvr|en i naro~ito uo~ljiv u postpubertetu. References BERBEROVI} LJ.& R.HAD`ISELIMOVI} 1982: Medical Antropology. Svjetlost, Sarajevo. BIELICKI T.& H. WALISZKO 1991: Urbanization-dependent gradients in stature among Polish conscripts in 1976 and 1986. American Yournal of Human Biology, 3: 419 – 424. BRODAR V. 1961: Fizi~ki razvoj {tudentov ljubljanske univerze v ~asu {tiriletnega {tudija. Biol. Vest. 8: 23 – 30. DOVE~AR F.& U. ARKO1978: Dinamika razvoja nekaterih telesnih parametrov glede na leto 1939/40. Glasnik ADJ, 15 (15): 61-69. FEDERAL BUREAU FOR STATISTICS – Sarajevo 1998: No : 052 – 0212 / 31. 03. 1998. GAVRILOVI} @.1972: Body Height and Weight of the First Ten Generations of Medical Technicians from Novi Sad. Medical Survey, 15: 3 – 4. GAVRILOVI} @.& V. BO`I} 1974: Some antropological karacteristics of the secondary school children. Zbornik Matice srpske za prirodne nauke, 46: 161 – 172. HAD`ISELIMOVI} R. (1988): Introduction into anthropogenesis theory. Svjetlost, Sarajevo: 25 – 53. HAD`ISELIMOVI} R.& S. LELO 1998: Bioanthropologic Practical Course. Faculty of Science and Mathematics, Sarajevo University, 35–55. Had`ihalilovi} J., Terzi} R., Ahmi} A., H. Halilovi} A., No`inovi} A., [ehi} A.: Secular trend... 43 stevilka 1_05.qxp 13.12.2005 12:32 Page 43 HAUSPIE R.C., VERCAUTEREN M. & C. SUSANNE 1997: Secular changes in growth and maturation: on update. Acta Pediatr Suppl , 423 : 20 – 7. HAUSPIE R.C., VERCAUTEREN M. & C. SUSANNE 1996: Secular changes in growth.Horm Res, 45 (Suppl. 2): 8-17. HIERNAUX J. 1975: Equality & Inequality of Races? [kolska Knjiga, Zagreb, 17 – 20. HISAFUMI M., KOSHI S., SHIGETAKA S.& M. MITSUNORI 1999: Bone Maturation the Secular Trend in Growth. Horm Res, 52: 125 – 130. IVANOVI} B. 1985: Ontogeny Development and Anthropologic Properties of Monte Negro Youths. CANU, Titograd, (1985): 13–52. KOVA~ I., EMILI H. & S. URBAN 1973: Body height and body mass of the school children of the island Pag today and before 30 years. Glasnik ADJ, 7: 123 – 135. KULENOVI} S. 1980: Tuzla Commune Population Structure and its Change Affected by recent economic Development. M. A. Degree Thesis. Faculty of Science and Mathematics, Beograd. MALINA R.M. 1990: Research on secular trends in auxology. Anthropologische Anzeiger, 48 : 209 – 27. MALINA R.M. 1979: Secular changes in size and maturity: causes and effects.Monogr. Soc. Res. Child. Dev., 179: 59- 102. NIKOLI}-DOVAT V. 1978: Somatic Growth and Development of a Child Institute of Mother and Child Public Health, Novi Sad: 19 – 25. NOVAKOVI} M. 1980: Growth and Pubertal Develpoment Male Youth From Tuzla’s Region. M.A.Degree Thesis. Medical Fakulty University of Tuzla, Tuzla. PREBEG @. 1978: Accelaration growthof the schoolchild and youth from Zagreb.Yugoslav. Akademy of Sciencees and Arts, »Rad« Knjiga 378, Zagreb. PREBEG @., JURE{A V.& M.KUJUND`I} 1995: Secular growth changes in Zagreb schoolchildren over four decades, 1951-91. Annals of Human Biology, 22 (2): 99 – 110. PREBEG @. 1997: Variations in Growth Patterns of Schooolchildren in Croatia over the Last Decade. In: Derek F.Roberts, Pavao Rudan, and [kari}-Juri} Tatjana (editors). Growth and Development in the Changing World. Croatian Anthropological Society, Zagreb. SANNA E., FLORIS G.& COSSEDDU G.G.1993: Secular trend in sardinian consripts drafted from 1979-1883 to 1983-1986. Antropologisher Anzeiger, 51: 225 – 232. STREL J., KOVA~ M., JURAK G., BRDNARIK J. & B. LESKO{EK 2001: Comparison of phisical development of school children between 1990 and 2000 on the basis of the data obtained from the sports educa- tional chart. Anthropological Notebooks,VII, No 1: 11-32. SUSANNE C. 1985: Living conditions and secular trend. J. Hum. Evol. 14: 357 – 70. [TEFAN~I} M., ARKO U., BRODAR V., DOVE~AR F., JURI~I} M. , MACAROL – HITI M., P. LEBEN – SELJAK & T. TOMAZO – RAVNIK 1996: Ocena telesne rasti in razvoja otrok in mladine v Ljubljani. Zdravstveno varstvo. Letnik 32, supl: 1. TANNER J. M.1962: Growth at adolescence. Blackwell: Oxford. Scientific Publications. p.313. TOMAZO – RAVNIK T. 1988: Secular trend in growth of Schoolchildren in Yugoslavia. Coll. Antropol. 12 (1): 121 – 125. TOMAZO – RAVNIK T. 1999: Body composition of youths in Slovenia. Abstracts. IV th International Anthropological Congress of Ale{ Hrdli~ka. WIERINGEN J.C. 1986: Secular growth changes. In : FALKNER F, TANNER JM. (eds.): Human growth, (3) 2 nd edn. New York: Plenum Press, pp. 307-312. WIERINGEN J.C. 1979: Secular Growth Changes – An Analysis of Developments in the Netherlands, 1850- 1978. Coll. Antropol. 3: 35 – 47. WEBER G., SEIDLER H., WILFING H. & G. HAUSER 1995: Secular change in height in Austria: an effect of population statification? Annals of Human Biology 22 (4): 277 – 288. Acta Biologica Slovenica, 48 (1), 2005 44 stevilka 1_05.qxp 13.12.2005 12:32 Page 44 Thinning of ’Golden Delicious’ apples with the combination of ethephon and CPPU Red~enje plodi~ev jablane sorte Zlati deli{es s kombinacijo etefona ter CPPU Matej STOPAR Agricultural Institute of Slovenia, Hacquetova 17, 1000 Ljubljana, Slovenia Abstract. A synthetic cytokinin forchlorfenuron (CPPU) 5 ppm was sprayed at the time of petal fall (PF) on eight years old ’Golden Delicious’/M.9 apple trees with the aim of enlarging the fruit growth. Since CPPU is not recognized as a thin- ning agent which increases the return bloom, a low dose of ethephon was sprayed on CPPU treated trees to enhance flower bud formation and increase flowering in the next season. Ethephon was applied in successive treatments (alone or in the combination with CPPU) 3 x 30 ppm or 5 x 50 ppm, starting at PF, and continued in week intervals. The single application of CPPU 5 ppm or the combination of ethephon 3 x 30 ppm with CPPU 5 ppm decreased the fruit set and enhanced the fruit growth to reach the commercial demand. The combination spraying of ethephon 5 x 50 ppm + CPPU 2 x 5 ppm resulted in a too strong thinning response while the yield of big- ger size fruit was not enhanced. The alone application of ethephon 3 x 30 ppm had no influence on the fruit thinning or fruit weight while the application of ethephon 5 x 50 ppm reduced the fruit set but, consequently, did not enhance the fruit growth. None of the treatments influenced the return bloom. Key words: apple thinning, CPPU, ethephon, alternate bearing Abbreviations: N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU), 2-chloroethylphosphonic acid (ethephon) Izvle~ek. Z namenom pove~anja velikosti plodov, smo {kropili osem let stare jablane ’Zlati deli{es/M.9’ s sinteti~nim citokininom forklorfenuronom (CPPU), v koncentraciji 5 ppm ob koncu cvetenja (KC) dreves. Ker CPPU ne izbolj{uje povratno cvetenje jablan, so CPPU tretiranim drevesom dodali {e etefon, kateri naj bi spodbudil nastanek cvetnega brstja ter tako izbolj{al cvetenje v naslednjem letu. Etefon je bil nane{en v ve~ zaporednih nanosih (sam ali v kombinaciji s CPPU), v tedenskih intervalih z za~etkom ob KC jablan in sicer 3x30 ppm ali 5x50 ppm. Samostojni nanos CPPU 5 ppm ali pa kombinacija etefona 3x30 ppm s CPPU 5 ppm je zmanj{ala rodni nastavek in pove~ala rast plodov kot zahteva komercial- ACTA BIOLOGICA SLOVENICA LJUBLJANA 2005 Vol. 48, [t. 1: 45-48 Sprejeto (accepted): 2005-09-27 stevilka 1_05.qxp 13.12.2005 12:32 Page 45 ni nivo pridelave. Kombinacija {kropljenj etefona 5x50 ppm + CPPU 2x5 ppm je povzro~ila premo~an osip plodi~ev, tako da se pridelek plodov ve~jega velikostnega razreda ni izbolj{al. Samostojni nanos etefona 3x30 ppm ni imel vpliva niti na red~enje plodi~ev niti na te`o plodov, medtem ko je etefon 5x50 ppm sicer zmanj{al rodni nastavek, kar pa posledi~no ni povzro~ilo pove~anja te`e plodov. Klju~ne besede: red~enje plodi~ev jablane, CPPU, etefon, izmeni~na rodnost Okraj{ave: N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU), 2-chloroethylphosphonic acid (etefon) Introduction Forchlorfenuron (CPPU), a synthetic cytokinin, has a strong influence on apple fruit growth if applied at petal fall (PF) or a few days later at the concentration below 10 ppm (GREENE 1989, GREENE 2001). Undesirable side effect, a reduced return bloom, was observed on ’Delicious’ and ’McIntosh’ apple trees after CPPU 5 ppm applications (CURRY & GREENE 1993, GREENE 1989). By contrast, an old thinner, ethephon, is known to enhance the return bloom of apple trees because its post bloom application mostly results in good flower bud induction (KNIGHt & BROWNING 1986). The application of ethephon mostly thins apple fruitlets while the growth of the remaining fruit, frequent- ly, does not respond with an enhanced growth rate (EBERT & BENDER 1986, LINK 2000, STOPAR 2000). In the experiment we tried to combine the positive effect of both growth regulators: applica- tion of ethephon a few times in a low concentration to enhance the return bloom and not to thin the fruitlets, and the use of CPPU for the enhancement of fruit growth on the high loaded trees. Material and methods Eight years old ’Golden Delicious’/M.9 apple trees were selected according to high bloom den- sity and homogeneous growth vigor. In the field trial a standard randomized block design with six replications and a single tree per plot were used. Successively, ethephon and CPPU were applied at weekly intervals and the first spraying of both agents started at petal fall (PF) time. The treatments were as follows: 1) Control – no thinning 2) Hand thin (done just after the June drop time) 3) CPPU 1x5 ppm = 5 ml Sitofex (SKW, Trostberk, Germany) / L water; 4) Ethephon 3x30 ppm = 0.06 ml Ethrel (Chromos, Zagreb, Croatia) / L water 5) Ethephon 5x50 ppm = 0.10 ml Ethrel / L water 6) Ethephon 3x30 ppm + CPPU 1x5 ppm; 7) Ethephon 5x50 ppm + CPPU 2x5 ppm; The spraying was done with a hand sprayer to the point of drip. No surfactant was used. When ethephon was combined with CPPU spraying, first, ethephon was applied and an hour later, after the leaves were dry, CPPU was applied. At maturity time the fruit was harvested, counted, weighed and graded into two size classes, < 68 mm and > 68 mm fruit diameter. Phytotoxic effect on trees was estimated a month after spraying with a visual scale (1 = no effect observed, 5 = suppressed shoot growth, bigger and wrinkled leaves with yellow green spots, more transparent crown). The return Acta Biologica Slovenica, 48 (1), 2005 46 stevilka 1_05.qxp 13.12.2005 12:32 Page 46 bloom was estimated visually next spring at bloom time using the scale 1-10 (1 = no flower clusters present on the trees; 10 = abundant flowering). During the experiment the trees received standard pest and disease management program. Data were subjected to statistical analysis using the statisti- cal program Statgraphics 5.0 (STSC, Rockwille, USA). Results and discussion The single PF application of CPPU 5 ppm significantly reduced the final fruit set (Table 1) and increased the fruit growth to satisfy the commercial demand for ’Golden Delicious’ apples (Table 2). When GREENE (2001) sprayed CPPU 8 ppm on ’McIntosh’ at PF he got no thinning effect while a week later spraying thinned the trees appropriately. STOPAR (1999) did not observe an enhanced fruit drop of small fruited apple cultivars ’Jonathan’, ’Elstar’ and ’Gala’ when CPPU 5 ppm was sprayed a week after PF. It looks that CPPU is not a thinner with a consistent fruitlet abscission response if sprayed either at PF or a week later. In this trial the return bloom after CPPU 5 ppm single spraying was not influenced too. Matej Stopar: Thinning of ’Golden delicious’ apples... 47 Treatment * Flower clusters (No./tree) Fruit (No./tree) Fruit (No./100 clusters) Return bloom (1-10)** 1) Control 191 a 142 cd 74 b 3,17 ab 2) Hand thin 219 a 101 bcd 47 a 1,83 a 3) CPPU 1x5 ppm 223 a 74 ab 33 a 4,0 ab 4) Ethephon 3x30 ppm 190 a 148 d 83 b 1,93 a 5) Ethephon 5x50 ppm 215 a 96 bc 44 a 4,38 ab 6) Ethephon 3x30 ppm + CPPU 1x5 ppm 221 a 76 ab 34 a 4,67 b 7) Ethephon 5x50 ppm + CPPU 2x5 ppm 196 a 42 a 22 a 4,00 ab Table 1: The number of flower clusters at the start of the experiment, the final fruit number and the return bloom of ’Golden Delicious/M.9’ apple trees after the application of thinning agents. Treatment * Yield (Kg/tree) Fruit > 68mm (Kg/tree) Fruit weight (g) Phytotoxicity (1-5)** 1) Control 16.0 b 5.5 ab 115 a 1,0 a 2) Hand thin 12.7 ab 8.4 ab 125 a 1,0 a 3) CPPU 1x5 ppm 10.9 ab 8.7 ab 161 b 2,2 b 4) Ethephon 3x30 ppm 15.5 b 4.5 a 106 a 1,0 a 5) Ethephon 5x50 ppm 10.8 ab 4.2 a 110 a 1,2 a 6) Ethephon 3x30 ppm + CPPU 1x5 ppm 12.7 ab 11.2 b 174 b 2,0 b 7) Ethephon 5x50 ppm + CPPU 2x5 ppm 7.2 a 6.8 ab 169 b 4,0 b Table 2: Total yield, yield of commercial (> 68 mm) fruit, mean fruit weight and the effect on phy- totoxicity after the application of thinning agents on ’Golden Delicious/M.9’ apple trees. stevilka 1_05.qxp 13.12.2005 12:32 Page 47 Application of ethephon 3x30 ppm did not affect the fruit set, yield or fruit growth of ’Golden Delicious’, while spraying of ethephon 5x50 ppm reduced the fruit set but did not enhance the fruit growth (Table 1,2). None of the ethephon alone treatments affected the return bloom in this trial sig- nificantly. The combination spraying of ethephon 3x30 ppm + CPPU 5 ppm reduced the fruit set and enhanced the fruit growth while the return bloom was not increased, i. e. all similar effects as those which happened after a single CPPU 5 ppm spraying. A stronger combination of both regulators (ethephon 5x50 ppm + CPPU 2x5 ppm) induced the overthinning of ’Golden Delicious’ trees. The fruit growth was enlarged but the yield of bigger size fruit was not increased because the fruit was not numerous. A very similar overthinning occurred when CPPU 5 ppm was combined with ethep- hon 3x70 ppm (PF + weekly intervals) on the cultivar ’Elstar’ (STOPAR 1998). In spite of overthin- ning the return bloom was not increased. This can be speculated as a diminishing effect of CPPU on the return bloom observed by GREENE (1989) and CURRY & GREENE (1993). A month after spraying the phytotoxic effect of treatments was estimated on trees. A week shoot growth, fewer but bigger leaves with green yellow spots and wrinkled surface were observed on CPPU treated crowns (Table 2). This phytotoxic effect was more pronounced on crowns on which CPPU was applied twice and the effect remaining on trees until harvest. Spraying of ’Golden Delicious’ with CPPU 5 ppm at PF thinned trees in our experiment, how- ever, bearing in mind CPPU trials reported by others no consistent thinning response was observed. If a weak phytotoxic CPPU 5 ppm effect is taken into consideration, no recommendation should be given to apple growers for the use of CPPU. For a more complete understanding of CPPU action the concentration and time response studies should be done on different apple cultivars. Literature CURRY E.A. & D.W. GREENE 1993: CPPU Influences Fruit Quality, Fruit Set, Return Bloom and Preharvest Drop of Apples. HortScience 28 (2): 115-119. EBERT A. & R.J. BANDER 1986. Influence of an Emulsifiable Mineral Oil on the Thinning Effect of NAA, NAAm, Carbaryl and Ethephon in the Apple Cultivar Gala Grown under the Conditions of Southern Brazil. Acta Horticulturae 179: 667-672. GREENE D.W. 1989: CPPU Influences ’McIntosh’Apple Crop Load and Fruit Characteristics. Hortscience 24 (1): 94-96. GREENE D.W. 2001: CPPU Influences Fruit Quality and Fruit Abscission of ’McIntosh’ Apples. Hortscience 36 (7): 1292-1295. KNIGHT J.N. & G. BROWNING 1986. Regulation of Conference Pear Cropping with Gibberelic Acid and Ethephon or Paclobutrazol. Acta Horticulturae 179: 337-342. LINK H. 2000: Significance of Flower and Fruit Thinning on Fruit Quality. Plant Growth Regulation 31: 17-26. STOPAR M. 1998: Introduction of New Environment Friendly Apple Thinning Compounds. Proceeding of the Conference Agriculture and Environment, Bled, p. 415-420. STOPAR M. 1999: NAA Thinning of ’Small-Fruited’ Apple Cultivars in Combination with CPPU. Phyton 39 (3): 85-90. STOPAR M., 2000. Comparison of the Most Frequently Used Apple Thinning Compounds for the Thinning of ’Jonagold’, ’Elstar’ and ’Golden Delicious’ Apples. Res. Rep. Biot. fac. UL – Agriculture 75 (1): 89-94. Acta Biologica Slovenica, 48 (1), 2005 48 stevilka 1_05.qxp 13.12.2005 12:32 Page 48