Acta Botanica 2-2015.indd ACTA BOT. CROAT. 74 (2), 2015 377 Acta Bot. Croat. 74 (2), 377-392, 2015 CODEN: ABCRA 25 ISSN 0365-0588 eISSN 1847-8476 DOI: 10.1515/botcro-2015-0026 Development of periphytic diatoms on different artifi cial substrates in the Eastern Adriatic Sea TINA NENADOVIĆ*1, TENA ŠARČEVIĆ2, HRVOJE ČIŽMEK2, JELENA GODRIJAN3, DANIELA MARIĆ PFANNKUCHEN3, MARTIN PFANNKUCHEN3, ZRINKA LJUBEŠIĆ1 1 University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, 10000 Zagreb, Croatia 2 »20.000 Leagues« Marine Explorers Society, L. Sorkočevića 3, 23000 Zadar, Croatia 3 Ruđer Bošković Institute, Centre for marine research, G. Paliaga 5, 52210 Rovinj, Croatia Abstract – The settling of diatoms as fouling organisms on a certain substrate is greatly infl uenced by substrate characteristics and the preferences of a diatom community and dia- tom species. A distinction among substrates can be made by analysing the specifi c abun- dance and composition of diatoms on different substrates. In this study, 11 different artifi - cial substrates were exposed to a marine environment for a period of 30 days. Abundance and taxonomic composition of periphytic diatoms was determined on each of the sub- strates and on shoots of the marine seagrass Posidonia oceanica. The aim was to compare diatom community structure on different newly colonized surfaces. On all surfaces exam- ined, periphytic diatoms were the pioneering organisms with differences in quantitative and qualitative composition on the different substrates. Taxonomic analysis of diatom communities on the substrates examined revealed 41 diatom taxa, with the dominant gen- era Cylindrotheca, Amphora, Nitzschia, Cocconeis and Navicula. Given that all the exam- ined artifi cial substrates were solid materials, differences in the abundance and species composition of diatoms found between the materials point to the substrates’ physical and chemical characteristics as a major infl uence on the fi nal settling of diatoms. Knowledge from investigating the settlement of fouling organisms on anthropogenic substrates can have future use in management of waste materials that end up in the marine environment. Key words: Adriatic Sea, anthropogenic materials, artifi cial substrates, diatoms, fouling, marine litter, periphyton Introduction Anthropogenic solid materials often end up in the marine environment and are com- monly known as marine litter. In their exposure to sea water, those materials undergo chem- * Corresponding author, e-mail: nenadovictina@gmail.com NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 378 ACTA BOT. CROAT. 74 (2), 2015 ical and biological conditioning known as the fouling process. Chemical conditioning pro- duces a molecular fi lm comprising compounds including glycoproteins, humic material, proteins, lipids, nucleic acids, polysaccharides, aromatic amino acids and/or unspecifi ed macromolecules (GARG et al. 2009 and the references therein), whereas biological condi- tioning supersedes with the accumulation of prokaryotic and eukaryotic unicellular organ- isms (RAILKIN 2004) incorporated in the matrix of extracellular polymers (DONLAN 2002). A newly formed conditioning matrix, or biofi lm, is mostly composed of bacteria and diatoms. Other unicellular organisms, like fl agellates, ciliates, yeasts and protozoans contribute less than 1% of the total cell count in the biofi lm (RAILKIN 2004). The presence of bacteria and unicellular algae in the biofi lm can enhance further colonization of the substrate by plants and animals (TOTTI et al. 2007 and the references therein). The initial colonizing biomasses, along with bacteria, are diatoms (WETHERBEE et al. 1998), eukaryotic microalgae, which constitute the periphyton community on immersed substrates. Diatom adhesion is mediated by the physico-chemical properties of the substrate and the diatom cell properties. The sur- face charge of diatom cells is dependent on their cell wall. Although the cell wall is sili- ceous and therefore negatively charged, it is covered with an organic layer of polysaccha- rides, proteins and glycoproteins (HECKY et al. 1973, STAATS et al. 1999), which allows cell surface potential to vary. Extracellular polymeric substances (EPS), secreted through nu- merous openings in the cell wall and the raphes is recognized as the extracellular adhesive which provides diatoms with the ability of permanent adhesion and motility on various substrates (WANG, et al. 1997, WUSTMAN et al. 1997). In the investigation of the diatoms’ cell surfaces and their ability of adhesion many techniques have been successfully used. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are tra- ditional and the most commonly used techniques preformed on dried samples. Since dia- toms form the bulk of initial colonizing biomass on immersed substrates, researchers have used artifi cial substrates to understand the preference of diatoms for a certain substrate (RO- DRIGUES and BICUDO 2001, DANILOV and EKELUND 2002, TANK and DODDS 2003, TOWNSEND and GELL 2005). The fi rst recorded study using an artifi cial substrate for periphyton settling was done by HENTSCHEL in 1915. The advantage of newly introduced artifi cial substrates in the marine environment is the opportunity to monitor initial development and the succes- sion of diatoms in the periphyton. Comparative research on nutrient concentration of pe- riphyton on living substrates (macrophytes, shells), epilithion (DANILOV and EKELUND 2002, KAHLERT and PETTERSSON 2002) and the periphyton on inorganic (glass, plastic and rock) and organic substrates (wood, leaves) showed that living and organic substrates act as ad- ditional source of nutrients for attached communities (DANILOV and EKELUND 2002, TANK and DODDS 2003). Some studies pertaining to the structural variations between artifi cial and natural substrates showed a signifi cant difference between artifi cial and natural substrates (TOWNSEND and GELL 2005) and others little (RODRIGUES and BICUDO 2001) or no structural difference (LANE et al. 2003). Also, the development of periphyton on man-made structures in the marine environment has become a widespread issue and the literature about periphy- ton development has been rapidly growing since the 1980s (BHOSLE et al. 1989, RAILKIN 2004, YEBRA et al. 2004, SHULTZ et al. 2011). Recent periphytic studies in the Adriatic Sea have focused on epiphytic diatoms of the northern Adriatic (MUNDA 2005) and the toxic bloom of benthic dinofl agellates Ostreopsis (TOTTI et al. 2007, PFANNKUCHEN et al. 2012), whilst in the middle Adriatic research is fo- cused on the ecology and taxonomy of periphytic diatoms in the estuary of rivers, like the River Zrmanja (BURIĆ et al. 2004, CAPUT et al. 2005). PERIPHYTIC DIATOMS ON DIFFERENT ARTIFICIAL SUBSTRATES ACTA BOT. CROAT. 74 (2), 2015 379 In this research, we examined the initial colonization of diatoms in the periphytic com- munity on a wide array of immersed artifi cial substrates with various physico-chemical properties. The abundance of diatoms on a newly formed biofi lm has been studied, as has the diatom genera composition. The seagrass Posidonia oceanica in a nearby meadow was also sampled to compare the abundance and composition of the diatom community from artifi cial substrates with an already established community on a natural substrate. The aim of this study was to determine the difference in abundance and composition of diatom com- munity on various artifi cial substrates and to discuss the difference in terms of diatom affi n- ity to a specifi c artifi cial material. The relevance of investigating the affi nity of diatoms as a major fouling community to a specifi c artifi cial material is to get an insight into the out- come of debris in the marine environment in order to systematically work towards alleviat- ing this initially negative impact on the marine environment. Materials and methods Study area The experiment was carried out in the Puntamika peninsula near Zadar, Croatia, coastal area of the Central Adriatic Sea (Fig. 1). The Adriatic Sea is an elongated basin, the north- ernmost part of the Mediterranean. The eastern coastal region is infl uenced by the ingoing current from the Ionian Sea, characterized by high salinity and low content of nutrients, and freshwater discharge from oligotrophic karstic rivers. The investigated station is situated in a region defi ned according to the physicochemical conditions as a region similar to the open waters of the middle Adriatic Sea (VIDJAK et al. 2012). Artifi cial substrates were exposed to the marine environment at a depth of 12 m for the period of one month (March 7th to April 6th 2012). Eleven different materials were used as artifi cial substrates: asbestos, painted iron, wood, concrete, glass, plastic, unpainted iron, rubber, ceramics, stone and aluminium. Materials used as substrates were of variable shapes and sizes and were acquired from the local waste deposit site. Description of the materials is as follows: glass – transparent glass used as a window, asbestos – plates used as roofi ng material, ceramic – smooth, glazed ceramic indoor tiles, rubber – rubber inner tube of a bi- cycle tire, wood – processed, smoothed wooden bar, plastic – hard non-transparent plastic Fig.1. Study area at Puntamika, Zadar, Croatia. NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 380 ACTA BOT. CROAT. 74 (2), 2015 plate, aluminium – fl at plate, iron – fl at plate, painted iron – painted fl at plate, rock – karstic rock, concrete – building block. Two replicates of each substrate were used in case of even- tual loss of one or more substrates. Substrates were submerged with the use of scuba diving to the depth of 12 m, which provided enough light for the development of periphyton, and protection from the infl uence of surge. Temperature of the sea while submerging was taking place was 11 °C, and 12 °C during the retrieval of the substrates. All substrates were placed on the seabed and arranged at 45º angle, leaning against a plastic pipe that followed the seabed at the location. The plastic pipe is an outlet pipe that has been there for a substantial amount of time. Plastic and rubber substrates were additionally anchored with materials (stone, glass jar) found at the location. Sampling After 30 days’ exposure, scuba divers retrieved one replicate of each substrate. Upon retrieval, the substrates were enclosed, each in a separate plastic bag to minimize the effect of the phytoplankton to the substrates. Upon retrieval, a 36 cm2 area of the mucous biofi lm covering the surface of the substrates was scraped from both the top and bottom sides of each substrate with a scalpel and brush. Scraped surface of each substrate was rinsed with distilled water into marked, wide-mouthed sample plastic vials and preserved in a 4% form- aldehyde solution. Two shoots of the seagrass Posidonia oceanica L. (Delile) from a nearby seagrass meadow were also sampled. The top parts of the shoots were cut, placed in a 0.5 L wide-mouthed sample container and preserved in 4% formaldehyde solution. In all, 22 samples were collected, 20 from the studied substrates and two shoots of P. oceanica. In two substrates, ceramics and concrete, sampling of the bottom side was not possible, due to roughness of the bottom surface of the materials and the inability to sample properly. Cell abundance analysis Fixed samples were stirred gently in plastic vials until they became homogeneous. Sub- samples were taken with a pipette and poured into 10 cm3 sedimentation chambers. Sam- ples containing Posidonia oceanica shoots were shaken vigorously and the surfaces of the shoots gently rubbed to remove the remaining periphytic diatoms and allowed to detach from the shoot surfaces into the suspension. After 24 hours of sedimentation, samples were analyzed in sedimentation chambers following the standard UTERMÖHL (1958) method us- ing an inverted microscope (ZEISS Axiovert 200, Zeiss GmbH). Due to the substantial amount of organic matter secreted by the cells and the small size of the cells, it was not pos- sible to determine diatoms under the light microscope, with the exception of Cylindrotheca closterium (Ehrenberg) Reinmann. C. closterium cell abundance was counted separately, due to its high dominance in the samples and the specifi city of the cells, which allowed easy determination. Taxonomic analysis For a more detailed taxonomic analysis using scanning electron microscopy (SEM), samples needed to be cleaned from cellular residue and organic matter. The cleaning pro- cess is based on the oxidation of organic matter with the method of strong acid oxidation. Samples were treated according to the von Stoch method (HASLE AND SYVERTSEN 1997) us- PERIPHYTIC DIATOMS ON DIFFERENT ARTIFICIAL SUBSTRATES ACTA BOT. CROAT. 74 (2), 2015 381 ing nitric acid (65%) and sulfuric acid (97%) in the quantity 1:1 and 3:1 in proportion to the sample volume respectively. The sample was stirred and heated to boiling point, which made the sample clear. After the sample was cooled, it was rinsed fi ve times with distilled water to remove the acid. After acid cleaning of the samples, samples were prepared for examination under the SEM. A drop of the cleaned diatom material was mounted on alu- minium stubs, air dried and gold coated with a sputter coater (S150A Sputter coater; Ed- wards Ltd., Crawley, UK). Observations were made with a Philips 515 SEM (FEI Co.). Diatoms were identifi ed to the genus level according to the classifi cation system of ROUND et al. (1990) using standard determination keys. Results Upon retrieval, after 30 days of exposure, a visible light brown macroscopic biofi lm could be observed on all submerged substrates with uniform distribution. There were no apparent visual differences between the biofi lms covering the substrates. Microscopic anal- ysis showed that the biofi lm consisted mostly of diatoms, with dinofl agellates, microscopic green algae (Chlorophyta) and brown algae (Phaeophyta) appearing sporadically. A marked difference in abundance of diatoms between different materials was recorded. Abundance of diatoms on artifi cial substrates The highest abundance of diatoms (6948 cells cm–2) was recorded on asbestos. Diatom abundance on other materials is as follows from greatest to least: painted iron (4622 cells cm–2), wood (4186 cells cm–2), concrete (3666 cells cm–2), Posidonia oceanica (2769 cells cm–2), glass (2266 cells cm–2), plastic (2261 cells cm–2), unpainted iron (1180 cells cm–2), rubber (1100 cells cm–2), ceramics (839 cells cm–2), stone (777 cells cm–2) and the lowest recorded abundance was on aluminium (216 cells cm–2) (Fig. 2). The mean abundance of diatom cells on studied substrates was 2569 cells cm–2. Artifi cial substrates were placed at an angle of 45 º on the sea bed, so the bottom sides of all the substrates, except concrete and ceramics, were sampled and abundance analyzed. The highest abundance on the bottom side of the substrates was on glass (2225 cells cm–2), followed by wood (1550 cells cm–2), rubber (1329 cells cm–2), plastic (653 cells cm–2), asbes- Fig. 2. Abundance of diatoms on top sides of examined substrates. NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 382 ACTA BOT. CROAT. 74 (2), 2015 tos (466 cells cm–2), unpainted iron (455 cells cm–2), stone (360 cells cm–2), aluminium (50 cells cm–2) and the least was on painted iron (46 cells cm–2) (Fig. 3). Mean abundance of diatom cells on bottom sides of studied substrates was 793 cells cm–2. After preliminary analysis of the samples using light microscopy, it was noted that the diatom Cylindrotheca closterium was the dominant species of diatoms present in all the samples. Therefore, in addition to the analysis of the abundance of all diatoms on investi- gated substrates, the abundance of the diatom C. closterium was analyzed independently. The highest abundance of C. closterium was recorded on wood (3091 cells cm–2), then painted iron (1161 cells cm–2), concrete (861 cells cm–2), plastic (569 cells cm–2), glass (424 cells cm–2), ceramics (383 cells cm–2), asbestos (238 cells cm–2), stone (161 cells cm–2), un- painted iron (140 cells cm–2), P. oceanica (137 cells cm–2), aluminium (16 cells cm–2) and the lowest on rubber (11 cells cm–2). The mean of C. closterium diatoms on the substrates studied was 599 cells cm–2. Abundance of C. closterium on the bottom sides of the sub- strates was also analyzed. The highest was on glass (132 cells cm–2), followed by wood (100 cells cm–2), stone (60 cells cm–2), plastic (50 cells cm–2), rubber (17 cells cm–2), asbes- tos (16 cells cm–2), painted iron (9 cells cm–2), unpainted iron (5 cells cm–2) and none was recorded on aluminium. The mean abundance of diatoms C. closterium on the bottom sides of studied substrates was 43 cells cm–2. Taxonomic analysis Taxonomic analysis revealed 41 diatom genera (Tab. 1), of which ten were identifi ed as planktonic genera sporadically present on several substrates. The dominant diatom species recorded on all substrates were Cylindrotheca closterium, identifi ed and counted with light microscopy, and diatom genera Amphora, identifi ed with SEM. The iron substrate had the highest diversity (20 taxa), the same as the diversity recorded in natural periphytic assem- blages on Posidonia oceanica (20 taxa). The lowest diatom diversity was recorded on plas- tic (4 taxa), concrete (4 taxa) and rubber (2 taxa) (Tab 1). Other diatom genera with fre- quent occurrence noted in the samples were Nitzschia (8 substrates + P. oceanica) (Fig. 4B), Cocconeis (7 substrates + P. oceanica) (Fig. 4C) and Navicula (7 substrates + P. oce- anica) (Fig. 4D). Fig. 3. Cylindrotheca closterium abundance on top sides of examined substrates. PERIPHYTIC DIATOMS ON DIFFERENT ARTIFICIAL SUBSTRATES ACTA BOT. CROAT. 74 (2), 2015 383 Tab. 1. Diatom diversity recorded on all examined substrates. Asterisk presents planktonic genera, P.o. – Posidonia oceanica, Ir. – iron, Gls. – glass, Al. – aluminium, Cer. – ceramics, P.Ir. – painted iron, As. – asbestos, St. – stone, W. – wood, Pls. – plastic, Crt. – concrete, Rub. – rubber. Substrate P.o. Ir. Gls. Al. Cer. P.Ir. As. St. W. Pls. Crt. Rub. Genus Amphora spp. × × × × × × × × × × × × Coconeis spp. × × × × × × × × Nitzschia spp. × × × × × × × × × Navicula spp. × × × × × × × × Haslea spp.* × × × × Entomoneis spp. × × × × × × Fallacia spp. × × × × Lyrella spp. × × × × Licmophora spp. × × × Striatella spp. × × × Striatella spp. × × × Grammathopora spp. × × × Tabularia spp. × × × Ardissonia spp. × × Campylodiscus spp. × × Campyloneis spp. × × Actinoptychus spp. × × Plagiogrammopsis spp. × Dimeregramma spp. × Microtabella spp. × Paralia spp. × × Opeophora spp. × × Pleurosygma spp.* × × Climaconeis spp. × × Diploneis spp. × Fragilaria spp. × Thalassiosira spp.* × Thalassionema spp.* × Auricula spp. × Parlibellus spp. × Toxarium spp. × Rophalodia spp. × Planktioniella spp*. × Astreromphalus spp.* × Bactreiastrum spp.* × NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 384 ACTA BOT. CROAT. 74 (2), 2015 Discussion Difference in quantitative and qualitative composition in the diatom community record- ed on the substrates investigated impliess the preference of diatoms for specifi c substrates. Quantitative values of diatom abundance were recorded on the top sides of all the sub- strates and shoots of the seagrass Posidonia oceanica from a nearby meadow. Materials of substrates used in this study vary in different physico-chemical properties, e.g. surface roughness, hydrophobicity, surface energy, solubility etc. With the substrates being placed at 45º angle, the top sides of the substrates had expectedly higher abundance of diatoms than the bottom side of the substrates due to the availability of the light diatoms use for Fig. 4. Scanning electron microscope microphotographs of dominant genera of diatoms found on va- rious substrates: A) Amphora sp. on glass substrate, B) Nitzchia sp. on ceramic tile substrate, C) Cocconeis sp. on aluminium substrate, and D) Navicula sp. on painted iron substrate. Substrate P.o. Ir. Gls. Al. Cer. P.Ir. As. St. W. Pls. Crt. Rub. Genus Membraneis spp.* × Chaetoceros spp.* × Delphineis spp.* × Mastogloia spp. × Berkeleya spp. × Psammodictyon spp. × Proschkinia spp. × Tab. 1. – continued PERIPHYTIC DIATOMS ON DIFFERENT ARTIFICIAL SUBSTRATES ACTA BOT. CROAT. 74 (2), 2015 385 photosynthesis. The interest in recording the diatom abundance on the bottom sides of sub- strates, where light is much more reduced, was in investigating whether any of the sub- strates acts as a nutrient source for the diatom community. The highest abundance of diatoms in this study was recorded on the asbestos substrate. High abundance of diatoms on asbestos suggests that this substrate is a favorable habitat for diatoms. Asbestos is composed of silicate fi bers, and as such, is chemically inert and resis- tant to biodegradation. Fibers could provide high protection from grazers and the space be- tween the fi bers can serve as a nutrient trap. Due to its porosity, it absorbs water and pro- longed exposure of asbestos in water leads to slow, progressive rinsing of metal and silicate compounds. Fibers of the most commonly used chrysotile asbestos (asbestos fi bers are di- vided into chrysotile and amphiboles) are coated with a brucite layer (MgOH2) which dis- solves relatively fast in water. The highly polar surface of chrysotile promotes adsorption of various organic and inorganic substances (SPEIL and LEINERVEBER 1969). As the brucite lay- er of chrysotile asbestos dissolves, magnesium ions (Mg2+) are rinsed into the surrounding medium as MgOH2 (CHISSICK 1985). High concentration of Mg2+, as shown by (SONG and LEFF 2006) in a study with Pseudomonas fl uorescens MIGULA 1895, can augment the pro- duction of exopolisaccharids (EPS) in bacteria, stabilize the structure of biofi lms and facili- tate further surface settlement (COSTERTON et al. 1995). Further investigation is needed to ascertain if asbestos used in this research releases Mg2+ to the surrounding water and what effect Mg2+ has on biofi lm formation. Painted iron had the second highest abundance of diatoms, after asbestos. The paint covering the surface of the iron makes the substrate’s surface very smooth, so the high abundance of diatoms on such a smooth surface is unexpected. In their study on microbial colonization, CHARACKLIS et al. (1990) recorded an increase in the extent of microbial colo- nization with substratum surface roughness. Common physico-chemical properties of metal substrata that could infl uence the settlement of organisms are their high surface energy and hydrophilicity. High surface energy of metal surfaces could promote adhesion, but is soon after immersion reduced by the adsorption of organic particles (KINLOCH 1990; CAILLOU et al. 2008). Hydrophilicity of metal surfaces, as shown in several previous studies (PEDERSEN 1990; BECKER and WAHL 1991; BECKER 1996), makes adhesion of diatoms more diffi cult as opposed to adhesion on hydrophobic surfaces. The composition of the paint covering with its specifi c, yet undetermined, chemical characteristics could have facilitated the adhesion and settlement of the diatoms and been responsible for the high abundance recorded on co- loured iron. Abundance of diatoms on wood was similar to that on painted iron. The high abundance of the diatom Cylindrotheca closterium on wood substrate greatly contributed to the high overall abundance of diatoms on wood. Our study showed that wood has proven to be a very favorable substrate for the diatom C. closterium. Wood can pose as a source of nutri- ents, as suggested in a study by VADEBONCOEUR and LODGE (2000) which concluded that the availability of nitrogen and phosphorus in the water column can depend on the level of wood degradation, and ZHANG et al. (2013) proposed that wood is a potential source of ni- trogen due to saprophyte domination. Furthermore, SCHOLZ and BOON (1993) showed that wood can be a substantial source of carbon for periphytic algae due to bacterial and fungal decomposition. A high abundance of diatoms was also recorded on a concrete substrate. Concrete was indicated to be an excellent substrate and habitat for the settling of periphytic organisms in NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 386 ACTA BOT. CROAT. 74 (2), 2015 »Guidelines for marine artifi cial reef materials« by LUKENS and SELBERG (2004). It is known that macroalgae can use some elements from the concrete (calcium, aluminium, iron and po- tassium) that they need for metabolism (BERTON 2004).The high roughness of concrete can trap detritus in the surface cracks which can serve as an additional source of nutrients (TANI- GUCHI AND TOKESHI 2004), as well as a protection from grazers (BERGEY and WEAVER 2004). Diatom abundance on Posidonia oceanica was similar to the mean abundance recorded on studied artifi cial substrates, but the diversity of diatoms was the same as the highest di- versity recorded on the unpainted iron substrate. Seagrass is a natural substrate for periphy- ton and the diatom community on shoots of seagrass was already a settled, more stable community. Macrophytes generally serve as a nutrient source for attached periphyton com- munity (CATTANEO and AMIREAULT 1992). Also, epiphytes on shoots of seagrass have better availability to light, as well as to nutrients from the water column and the substrate (HUTCHINSON 1975). Additionally, macrophytes as an elastic substrate can sustain wave en- ergy and reduce turbulent effects on the biofi lm surface, which could promote biofi lm for- mation (PFANNKUCHEN et al. 2012). Glass and plastic substrates showed very similar diatom abundance, around the mean value. Glass and plastic panels have been widely used as artifi cial substrates for the settle- ment of diatoms in both marine and fresh water environments by many researchers (COOK- SEY et al. 1984, WAHL and MARK 1999, CAPUT et al. 2005, NAYAR et al. 2005, WEBSTER and NEGRI 2006, REISSER et al. 2014). Although both materials are inert in the marine environ- ment, glass and plastic have different physico-chemical properties. Glass is a high-energy hydrophilic surface, while plastic is a low-energy hydrophobic surface, and as reported by many studies, diatoms adhere more successfully to hydrophobic surfaces (FLETCHER 1988, BECKER and WAHL 1991, BECKER 1996). The shared property of both substrates is the smoothness of the substrate, which could be the cause of the similar diatom abundance. The unpainted iron substrate showed low diatom abundance, but the highest diatom di- versity. The hydrophilicity of iron makes the adhesion of diatoms on metal surfaces diffi - cult. This is also attributed to the low surface energy caused by organic layer, which is ef- fective in preventing adhesion (TOWNSIN and ANDERSON 2009) as mentioned before. Metals in sea-water form hydroxides biologically unavailable for uptake by algae (LEWANDOWSKA and KOSAKOWSKA 2004). Most studies of diatoms, and biofi lm in general, on metal surfaces have investigated diatom biofi lms on hydrodynamic drag on vessels (BOHLANDER 1991, SHULTZ et al. 2011) and the biofi lm development on stainless steel surfaces (SCHNEIDER 1996, LANDOULSI et al. 2011). Diatom abundance on a rubber substrate was as low as that on unpainted iron. Although rubber is inert in the marine environment, some research has focused on leaching of heavy metals and organic compounds from rubber to surrounding water. Heavy-metal leaching in seawater could be effective in preventing the colonization of the substrate by fouling organ- isms, as reported by JELIĆ-MRČELIĆ (2006) in tests with heavy-metal leaching antifouling (AF) coatings. Zinc, as a major toxicant that rubber leaches into the water environment, was identifi ed by COLLINS et al. (1995) and GUALTIERI et al. (2005), but according to their study, the amount of zinc being released in that way does not have any signifi cant effect on most marine organisms. The settling of fouling communities on rubber substrates should be stud- ied in more detail, since rubber is one of the most common materials used for artifi cial reefs. Also, the disposal of rubber tires has become a widespread issue as large amounts of car tires are being disposed of in the marine environment (personal observation), especially for im- PERIPHYTIC DIATOMS ON DIFFERENT ARTIFICIAL SUBSTRATES ACTA BOT. CROAT. 74 (2), 2015 387 plementing artifi cial reefs and often without any prior investigation of the infl uence of tires on the marine environment and their suitability for the settling of marine fouling organisms. The extremely smooth surface of the glazed ceramic tile could be the reason for low diatom abundance. The results of MURDOCK and DODDS (2007) in their study with benthic microalgae showed higher abundance of algae on unglazed than on glazed ceramic tiles. Studies by CHARACKLIS et al. (1990), investigating the adhesion of bacteria on surfaces of different roughness, and WOODS and FLETCHER, (1991), investigating adhesion of diatoms on rough and smooth surfaces, came to the same conclusion – that the adhesion for both bacteria and diatoms was more successful on rougher surfaces because of the bigger sur- face for attachment in comparison with smooth surfaces. Low abundance on stone substrata was surprising given that stone is a natural substrate for the periphyton, and has a rough surface texture. Periphyton on stone substrates can ac- quire nutrients from the surrounding water column or microbial regeneration inside the pe- riphyton matrix (STEVENSON and GLOVER 1993). Aluminium substrates had the lowest diatom abundance of all the substrates investigat- ed. The very smooth surface of aluminium can make adhesion of diatoms diffi cult and fa- cilitate detachment of cells from the surface. Also, it is possible that aluminium and alu- minium oxide could have a toxic effect on periphytic organisms. Several studies showed that aluminium dissolved in sea-water inhibits the growth of some planktonic species by reducing the amount of available dissolved phosphorus (DICKSON 1978, DRISCOLL 1985) or with direct toxic effects (FOLSOM et al. 1986). The interest in recording the abundance of bottom sides of the substrates where light is much more reduced was in investigating whether any of the substrates acts as a nutrient source for the diatom community. The bottom side of the glass substrate had the highest abundance of diatoms. The transparency of the glass plate used in this study made photo- synthesis available to the diatom community on the bottom side of the substrate. High dia- tom abundance on the bottom side of wood substrate could be attributed to wood being a nutrient source for the periphyton community, as mentioned before. Rubber substrate showed high abundance of diatoms and was the only substrate that showed higher abun- dance of diatoms on the bottom side of the substrate. The reason for this could be in the method the rubber substrate was anchored to the sea bed. The rubber used in this research was a black bicycleinner tube anchored around a glass jar found on the location, so the bot- tom side of the rubber was in direct contact with the glass jar from which the already estab- lished diatom community could migrate to the rubber substrate. Plastic, asbestos, unpainted iron and stone showed similar low abundances, that can be explained by insuffi cient light availability. Aluminium and painted iron had extremely low abundances, which could be attributed to the antimicrobial effects of metals (JAIN 1990), as well as to the low light inten- sity on the bottom side of the substrates. Abundance of the diatom Cylindrotheca closterium, due to its specifi city and high abun- dances in the preliminary examination of substrates, was recorded for the top and bottom sides of all substrates. By far the highest abundance was on wood substrate, followed by painted iron, concrete, plastic, glass, ceramics, asbestos, P. oceanica, unpainted iron, stone, and the the lowest was on aluminium and rubber. The abundance of the bottom side followed the same trend, with the exception of the glass substrate, which showed the highest abun- dance of C. closterium due to its transparency and the availability of light. It can be con- cluded that the wood substrate is the most favorable substratum for the diatom C. closterium. NENADOVIĆ T., ŠARČEVIĆ T, ČIŽMEK H., GODRIJAN J., MARIĆ PFANNKUCHEN D., et al. 388 ACTA BOT. CROAT. 74 (2), 2015 This study recorded Cylindrotheca closterium and the genera Navicula, Nitzschia, Coc- coneis and Amphora as dominating diatoms in the periphyton community on the artifi cial substrates studied. It has been shown that pennate diatoms dominate diatom communities in biofi lms (PATIL and ANIL 2005, and included citations) with frequently identifi ed pennate genera Navicula, Nitzschia, Cocconeis, Amphora (RAILKIN 2004), as was recorded in this study. The majority of studies showed the dominance of pennate genera Amphora, Navicu- la, Nitzchia (KHATOON et al. 2007) and Cylindrotheca (MOLINO et al. 2009; DOBRETSOV and THOMASON 2011; BRIAND et al. 2012) on different artifi cial substrates as well as on fouling release coated substrates (CASSÉ and SWAIN 2006; ZARGIEL et al. 2011). The recorded genera of diatoms in this study are therefore typical periphytic diatoms in biofi lms. In conclusion, this study recorded a qualitative and quantitative difference of diatom abundance in periphyton community among different artifi cial substrates. It was proven that the settlement of diatom community on a specifi c substrate is dependent on light avail- ability, surface roughness and properties. We hypothesize that some substrates, like wood and concrete, serve as a nutrient source to the periphyton community. Hydrophobicity of the substrates was not shown to have a major infl uence on diatom settlement. As mentioned in the discussion, there have been many efforts at fi nding favorable artifi - cial reef materials that promote the biodiversity of marine organisms. Development of pe- riphyton is the fi rst step towards the settlement of higher organisms, and, provided with a suitable substrate, the biodiversity could be enhanced. 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