158 ACTA BOT. CROAT. 80 (2), 2021 Acta Bot. Croat. 80 (2), 158–168, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2021-016 ISSN 0365-0588 eISSN 1847-8476 Biodiversity and seasonal distribution of benthic diatom assemblages as an indicator of water quality of small karstic river in Bosnia and Herzegovina Anita Dedić1*, Dubravka Hafner2, Ana Antunović1, Jasmina Kamberović3, Svjetlana Stanić-Koštroman1, Martyn G. Kelly4 1 University of Mostar, Faculty of Science and Education, Department of Biology, Matice hrvatske bb, BiH-88000 Mostar, Bosnia and Herzegovina 2 Bartulovići 4, HR-20357 Blace, Croatia 3 University of Tuzla, Faculty of Natural Sciences and Mathematics, Department of Biology, Urfeta Vejzagića 4, BiH- 75000, Tuzla, Bosnia and Herzegovina 4 Bowburn Consultancy, 11 Monteigne Drive, Bowburn, Durham DH6 5QB, UK Abstract – The aims of this paper were to describe seasonal changes in the qualitative and quantitative composition of diatom taxa and the potential application of benthic diatoms for ecological status evaluation. Diatom indices (IPS and TI) were calculated from data from three different locations along a longitudinal profile of the Bunica, a small karstic river in Bosnia and Herzegovina. A total of 147 taxa were recorded in 12 samples. The most common taxa were Meridion circulare (Greville) C.Agardh and Ulnaria ulna (Nitzsch) Compère. Physical and chemical analyses showed low concentrations of nutrients, good oxygenation, typical pH for carbonate bed/origin and generally oligotrophic conditions and high ecological status. All sites had similar physico-chemical conditions and there were only few sea- sonal differences. Ordination of the diatom data showed that samples showed neither longitudinal nor seasonal pat- terns. Median value for IPS (16.8) and for TI (7.3) can be possible ‘‘expected’’ values for ecological status assessment for small karstic rivers in the Mediterranean region. We propose the use of the phytobenthos Intercalibration Common Metric (pICM – an index that combines the IPS and TI) as a national metric for countries developing WFD diatom methods at a late stage. One situation is described, and a solution, which is potentially transferable to other locations in Bosnia and Herzegovina and also to other countries facing similar challenges. Keywords: Bacillariophyceae, diatom indices, ecological status, Mediterranean river, phytobenthos, pICM Introduction The Water Framework Directive (Directive 2000) led to the adjustment of existing assessment systems for water quality and encouraged the design of new classification sys- tems for the ecological status of rivers. The new systems in- cluded new approaches to the definition of reference condi- tions (Pardo et al. 2011, 2012, 2018), the establishment of national and European river typologies, the definition of ecological status class boundaries, and the mandatory in- tercalibration of boundaries between European countries (Van de Bund 2009). Ecological status assessment has to be type-specific as streams have different biological communi- ties and reference conditions due to their physical and mor- phological attributes, such as stream size, altitude, catch- ment geology, etc. Benthic diatoms are one group of organisms widely used as ecological indicators in water quality and ecological status assessment and monitoring and are widely used as proxies for “phytobenthos” in WFD assessments. Many indices based on species-specific sensitivities and tolerances have been developed to infer the environmental conditions in streams and rivers. The WFD requires that methods are ex- pressed as Ecological Quality Ratios (EQRs), defined as the “observed” (O) value of a metric divided by the value “ex- pected” (E) at reference condition (O/E). In this study two * Corresponding author e-mail: anita.dedic@fpmoz.sum.ba BENTHIC DIATOMS OF SMALL KARSTIC RIVER ACTA BOT. CROAT. 80 (2), 2021 159 diatom indices; IPS (Indice de polluo-sensibilite, CEMAGREF 1982), and TI (Trophic Index, Rott et al. 1999) were used to estimate ecological status in a small karstic river in Bosnia and Herzegovina (BH). It presents the relationship between measured water quality variables in the Bunica River and values of diatom indices. Two diatom metrics (IPS and TI) were compared and then combined into the phytobenthos In- tercalibration Common Metric (pICM, Kelly et al. 2009). Also, various diversity indices are used to determine the distribution of benthic diatoms. Diversity index is a statisti- cal method which is planned to evaluate the variety of a data group consisting of different types of components. Number of existing species (richness), distribution of indi- viduals equally (evenness) and total number of existing in- dividuals underlie the basis of diversity indices. Three di- versity indices (the Shannon Diversity Index, Simpson Diversity Index, Margalef Diversity Index) and one even- ness index (Pielou’s Evenness Index) were used in this paper. The purposes of this paper are: to present the list of ben- thic diatom assemblages; to assess the seasonal variations and ecological status of the river; to test the use of two dia- tom indices as tools for estimating the ecological status of small karstic rivers; and to explore the potential for using the pICM as a national metric for countries such as BH which are developing WFD-compatible phytobenthos methods at a relatively late stage. Material and methods Study area The Bunica River is a limestone karstic river in the south of Bosnia and Herzegovina and is a tributary of the Buna River, itself part of the Neretva River basin (Fig. 1). In terms of geology and geomorphology the Bunica River is part of the Dinaric karst or Dinaric carbonate platform – external Dinarides (Milanović 2006). Thick layers of carbonate- formed long carbide precipitation characterize this area. The dominant clasts of the sediment are cobbles and sand. The siphon spring of the Bunica River is the underground extension of the flow of the Zalomka River (Fig. 1). The si- phon is 73 m deep and ranks among the largest sources of the Dinaric karst (Milanović 2006). The minimum and maximum discharges registered for the Bunica Spring were 0.72 and 207 m3 s–1 (Milanović 2006). It is located in an area with a Mediterranean climate, characterized by long, hot summers and rainy winters with rare occurences of snow (Galić 2011). The average annual air temperature is 14.6 °C. The highest daily average temperature is 23.2 °C in July and the lowest below 5.8 °C in January (data for the city of Mostar, for the period 1971–2000, recorded by Meteorological and Hydrological Service of Bosnia and Herzegovina). The total annual precipitation is about 1515 mm. This area has ap- proximately 2291 hours of sunshine per year. Average rela- tive humidity is 69. So far, diatoms were studied only in the spring area of the Bunica River, revealing high diversity of taxa on artifi- cial (glass slides) and natural substrates (Dedić 2015, Dedić et al. 2015). Sampling and analyses The ecological river typology in Bosnia and Herzegovina was developed according to System B of the WFD Annex II, which allows any natural environmental parameter-influ- encing communities to be included. A total of 216 water bod- ies of surface waters have been identified in the Adriatic Sea river basin district in the Federation of BH, specifically 211 running waters, four (4) stagnant waters and one (1) coastal water body (Mrđen et al. 2018). Within this typology the Bu- nica River belongs to type 12, subundertype 12a, small and medium lowland rivers on limestone sediment. For this study, a total of 12 benthic diatom samples (three sampling locations: the source of the river, the middle course, and the lower course, each each once per season: spring – May, summer – July, autumn – October in 2013, and winter in January 2014) were collected by scraping the biofilm from rock surface, applying the the standard procedure of scrap- ing sludgy material from the rock surface while making sure that the total surface of all rocks is about 500 cm2 (CEN 2003). The upper surface of each rock was scratched with a scalpel and carefully scrubbed with a toothbrush. The sam- ples were fixed with 4% formaldehyde and stored in appro- priately labelled bottles. In the laboratory, samples were treated with concentrated sulphuric acid, potassium-per- manganate and oxalic acid (Krammer and Lange-Bertalot 1986). The cleaned suspension was used to make permanent diatom microscope slide preparations. The composition and relative abundance of diatoms were estimated at 1000× mag- nification, using a Carl Zeiss Jena light microscope. At least 400 valves were counted and identified. Identification and nomenclature were based on the relevant scientific literature and keys: Krammer and Lange-Bertalot (1986–1991), Lange- Bertalot (2001), Krammer (2000–2003), Krammer and Lange-Bertalot (2004). The nomenclature of taxa follows AlgaeBase (Guiry and Guiry 2020). Physico-chemical parameters (pH, electric conductivity (EC), dissolved oxygen (DO), oxygen saturation (SA) and Fig. 1. Sampling sites marked with numbers 1, 2 and 3 along the studied Bunica River, tributary of the river Buna in Bosnia and Herzegovina. DEDIĆ A., HAFNER D., ANTUNOVIĆ A., KAMBEROVIĆ J., STANIĆ-KOŠTROMAN S., KELLY M. G. 160 ACTA BOT. CROAT. 80 (2), 2021 temperature of water (T)) were measured at the same time that diatom samples were collected using a WTW probe (Wissenschaftliche-Technische Wersättem GmbH & Co. KG-Weilheim). Water samples were also taken and analysed by standard spectrophotometric methods in in the Labora- tory of Public Health Mostar to current norms. The follow- ing chemical parameters were analyzed: dissolved oxygen (DO), chemical oxygen demand (COD), nitrate (NO3–), am- monium (NH4+), total nitrogen (TN), total phosphorus (TP), orthophosphate (PO43–) and silica (SiO2). Diatom in- dices were calculated using OMNIDIA GB 5.3 software ( Lecointe et al. 1993). Indices taken into account for the as- sessment of water quality are those with the highest propor- tion of species used in the calculation. Values of indices in OMNIDIA software range from 1 to 20, indicating the quality range from polluted to clean water. The following indices were used: IPS (CEMAGREF 1982) and TI (Rott et al. 1999). We estimated diatom diversity using Shannoń s (H´), Simpsoń s (1-lambda), Margalef ś (d) diversity indices and Pielou ś Evenness Index (J). Indices were calculated with the PAST software (Hammer et al. 2001). Chemical data were processed using principal compo- nent analysis (PCA) to obtain different water quality groups. The variables used in these analyses were pH, electrical con- ductivity, dissolved oxygen, oxygen saturation, water tem- perature, chemical oxygen demand, ammonium, nitrate, total nitrogen, total phosphorus, orthophosphate and silica. Diatom data were analyzed using Primer v.6 (Clarke and Gorley, 2006): hierachical group average clustering, SIMPROF test, Principal Coordinates Analysis (PCO), to represent the distribution of the communities; SIMPER analysis (SIMilarity PERcentage) to estimate the degree of similar- ity within and among different groups (i.e., reference vs non-reference), and to estimate the individual contribution and the importance of each taxon to the global similarity among groups. These analyses were based on the Bray- Curtis similarity matrix which combines information on the rela- tive abundance of species and faithfulness of the occurrence of species abundances in particular groups. The phytobenthos Intercalibration Common Metric (pICM) was conceived by Kelly et al. (2009) as a means of converting national assessment metrics to a common scale. It is calculated as the average of EQRs (ecological quality ratios) computed using the Indice de Polluosensibilité Specifique (IPS, CEMAGREF 1982) and the Trophic Index (TI: Rott et al. 1999). The pICM is defined as the average of two EQRs: ICM = (EQRIPS + EQR TI )/2. The pICM metric responds along a long gradient of in- organic and organic enrichment (Kelly et al. 2009, 2012). For this study we adopted the “expected” values of 16.8 (IPS) and 7.3 (TI) used for streams similar to the karstic streams in this study during the Mediterranean phytobenthos inter- calibration exercise (Almeida et al. 2014). We also used the average position of the high/good and good/moderate eco- logical status class boundaries (0.9 and 0.69) established during this study as a starting point for understanding the ecological condition of these streams in comparison to oth- ers in the Mediterranean basin. Both metrics were calculated using Omnidia version 5.3 (Lecointe et al. 1993) and converted to EQRs by dividing the observed value by the ‘expected’ value. The pICM was then transformed against national metrics and national EQRs for High/Good and Good/Moderate status boundaries convert- ed to equivalent values of pICM. Results Physico-chemical and chemical variables per seasons The main abiotic variables measured in the Bunica River are shown in Tab. 1. Nutrient concentrations are low, the water is well-oxygenated and pH is typical for water of Tab. 1. Main abiotic variables in the Bunica River measured in three sites along a longitudinal profile of the Bunica River once per season (spring, summer, autumn and winter in the period from 5th May 2013 to 9th January 2014). Min. – minimum value, Max. – maximum val- ue, Avg. – average value, SD – standard deviation, T – temperature, EC –conductivity, DO – dissolved oxygen, SA – oxygen saturation, COD – chemical oxygen demand, NH4+ – ammonium, NO3– – nitrate, TN – total nitrogen, SiO2 – silica, TP – total phosphorus, PO43– – orthophosphate. Parameter/ Station Bunica 1 (n=4) Bunica 2 (n=4) Bunica 3 (n=4) Min. Max. Avg. SD Min. Max. Avg. SD Min. Max. Avg. SD T (°C) 11 14.2 12.8 1.4 11.4 15.5 13.7 1.7 11.4 17 14.6 2.3 pH 7.6 8 7.8 0.17 7.7 7.9 7.8 0.05 7.8 7.9 7.8 0.04 EC (µS cm–1) 313 403 372.5 40.4 314 406 371.5 39.8 312 406 369.2 40.3 DO (mg L–1) 9.65 12.25 11.28 1.16 11.2 12.7 11.88 0.66 11.43 13.3 12.2 0.84 SA (%) 90.1 119.3 106.9 13.4 90.1 127.9 107.2 15.6 103.4 132 120.8 12.3 COD (mg L–1) 0.77 1.28 1.02 0.23 1.02 3.58 2.07 1.17 0.89 2.43 1.47 0.66 NH4+ (mg L–1) 0 0 0 0 0 0.003 0.0007 0.0015 0 0.003 0.0007 0.001 NO3– (mg L–1) 0.18 0.45 0.35 0.12 0.29 0.49 0.37 0.08 0.27 0.43 0.36 0.067 TN (mg L–1) 0.30 0.61 0.47 0.13 0.37 0.55 0.47 0.07 0.37 0.52 0.45 0.062 SiO2 (mg L–1) 2.68 4.8 3.63 0.97 3.10 4.71 4.04 0.68 3.14 4.20 3.86 0.48 TP (mg L–1) 0.006 0.011 0.008 0.002 0.004 0.01 0.007 0.0009 0.006 0.008 0.0066 0.0009 PO43– (mg L–1) 0.002 0.005 0.0035 0.00 0.002 0.003 0.0026 0.0007 0 0.005 0.0018 5.13 BENTHIC DIATOMS OF SMALL KARSTIC RIVER ACTA BOT. CROAT. 80 (2), 2021 161 carbonate bed/origin and generally oligotrophic conditions. Water quality corresponds to natural status without anthro- pogenic influence, following Anonymous (2014) and should support high ecological status. Differences between sites along the Bunica River, and fluctuations among the seasons were not pronounced. Wa- ter temperature ranged from 11–17 °C (Tab. 1) and showed relatively low values for all the seasons. The lowest seasonal fluctuations were recorded at the spring. Dissolved oxygen concentration (9.65–13.3 mg L–1) and saturation (90.1–132%) (Tab. 1) were high at all sites and in all seasons. Conductiv- ity ranged from 312 to 406 µS cm–1 (Tab. 1) with the lowest values in spring, and the highest in winter. All sites had slightly alkaline pH values (7.6 – 8) (Tab. 1). The most sig- nificant positive correlation was achieved among total ni- trogen and silica (r = 0.9, P < 0.01), and negative correlation was found for total nitrogen and oxygen saturation (r = 0.677, P < 0.01). Multivariate analysis of the main chemical variables (PCA) in three dimensions accounted for 74.2% of the variance. The first three axes (PC1, PC2 and PC3) ac- counted for 45.4%, 18.9%, and 9.9% of the variance, respec- tively (Tab. 2). The most important parameters for the PCA axis 1 were TN and NO3–, with the coefficients in the linear combinations of variables of 0.400 for TN, and 0.374 for NO3–. The variables that weighted most for ordination for the PCA axis 2 were the COD and PO43– (coefficients in the linear combinations of variables: –0.477 and 0.432, respec- tively). The first axis differentiated spring and summer sam- ples with low scores on PC1 from autumn and winter sam- ples with high PC1 scores (Fig. 2). Analysis of benthic diatoms In total, 147 diatom taxa from 43 genera were identified in the 12 samples from the Bunica River (Tab. 3). The gen- era with the highest number of taxa were Gomphonema (23 taxa), Navicula (18), Cymbella (10), Nitzschia (10) and Cocconeis (7). Meridion circulare (Greville) C.Agardh and Ulnaria ulna (Nitzsch) P.Comparé, recorded in all samples, followed by Cocconeis placentula Ehrenberg, C. euglypta (Ehrenberg) Grunow, C. lineata Ehrenberg, Diatoma vulgaris Bory de Saint-Vincent, D. vulgaris var. capitulata Grunow, D. vulgaris var. producta Grunow, Encyonema ventricosum (C.Agardh) Grunow, E. silesiacum (Bleisch) D.G. Mann, Gomphonema olivaceum (Hornemann) Brébisson, Melosira varians C.Agardh, Navicula tripunctata (O.F. Müller) Bory de Saint-Vincent, Rhoicosphenia abbreviata (C. Agardh) Lange-Bertalot and Navicula cryptocephala Kützing. All sampling stations had a similar species rich- ness in all seasons. The number of taxa at Bunica 1 (spring area) ranged from 30 (in winter and summer) to 43 (in au- tumn), from 33 (spring) to 53 (autumn) for Bunica 2 (middle part of the river) and 40 (summer) to 49 (autumn) for Bunica 3 (the mouth of river). Values of Diversity and Evenness Indices per samples are shown in Tab. 4. Shannon’s Diver- sity Index ranged from 1.80 to 2.75, Simpson’s Diversity In- dex from 0.69 to 0.90, Margalef ’s Diversity Index from 4.50 to 8.42 and Pielou’s Evenness Index from 0.20 to 0.41. The higest values of indices were recorded for the middle part of river (Bunica 2), mostly in summer (Tab. 4). The lowest val- ues were in spring but for different locations (Tab. 4). SIMPER analyses showed high disimilarity among the seasons. The dissimilarities among the samples in different seasons ranged from 60.83% (spring-summer) to 70.24% (autumn-winter). The spring group showed 36.8% similar- ity, and was characterized by Melosira varians (MVAR), Diatoma vulgaris var. capitulata (DVCA), Meridion circulare (MCIR) and Gomphonema olivaceum (GOLI). The summer group showed 37.77% similarity, characterized by Encyonema ventricosum (EVEN), Navicula tripunctata (NTPT), and Cocconeis placentula (CPLA). Autumn samples showed the lowest similarity, 35%, with C. euglypta (CPLE), Achnanthes sp. (ACSP) and M. circulare (MCIR) as the most typical spe- cies. Winter samples had 35.9% similarity and were charac- terized by M. circulare (MCIR), Ellerbeckia arenaria (EARE) and C. placentula (CPLA). Similar results were obtained for the analysis of longitudinal gradient/locations with dissim- ilarities ranging from 64.79% between the first and second sites to 68.6%, between the second and third locations. Fig. 2. Principal component analysis (PCA) ordination diagram performed on the environmental parameters for all sampling sta- tions (1, 2, 3) in the Bunica River during four periods (a-spring, b-summer, c-autumn, d-winter) and overlapped with Pearson correlation vectors with PC1 and PC2 axes (r > 0.4). Abbrevia- tions: EC – conductivity, T – temperature, NH4+ – ammonium, NO3- – nitrate, TN – total nitrogen, SiO2 – silica, PO43- – ortho- phosphate, TP – total phosphorus, COD – chemical oxygen de- mand, DO – dissolved oxygen, Sat. – oxygen saturation. Tab. 2. Characteristic values of all five axes of principal compo- nent analysis (PCA) with percentage variance for a total of 12 in- vestigated parameters. PCA 1 2 3 4 5 Eigenvalues 5.44 2.27 1.19 1.04 0.872 % variation 45.4 18.9 9.9 8.7 7.3 Cumulative % variation 45.4 64.3 74.2 82.9 90.1 DEDIĆ A., HAFNER D., ANTUNOVIĆ A., KAMBEROVIĆ J., STANIĆ-KOŠTROMAN S., KELLY M. G. 162 ACTA BOT. CROAT. 80 (2), 2021 Tab. 3. Identified diatom taxa with their maximum abundances (%) in a total of 12 samples in the Bunica River. Taxon Max (%) Achnanthes inflata (Kützing) Grunow 8.33 Achnanthes sp. 83.33 Achnanthidium minutissimum (Kützing) Czarnecki 16.67 Achnanthidium pyrenaicum (Hustedt) H.Kobayasi 41.67 Amphipleura pellucida (Kützing) Kützing 8.33 Amphora ovalis (Kützing) Kützing 41.67 Amphora pediculus (Kützing) Grunow 58.33 Amphora sp. 8.33 Aneumastus sp. 8.33 Aneumastus tuscula (Ehrenberg) D.G.Mann et A.J.Stickle 25 Aulacoseira italica (Ehrenberg) Simonsen 41.67 Brachysira microcephala (Grunow) Compère 16.67 Brebissonia lanceolata (C.Agardh) Mahoney et Reimer 16.67 Caloneis bacillum (Grunow) Cleve 8.33 Caloneis placentula Ehrenberg 8.33 Caloneis silicula (Ehrenberg) Cleve 8.33 Caloneis ventricosa var. truncatula (Grunow) Meister 33.33 Campylodiscus noricus Ehrenberg ex Kützing 8.33 Cocconeis euglypta Ehrenberg 91.67 Cocconeis lineata Ehrenberg 91.67 Cocconeis pediculus Ehrenberg 66.67 Cocconeis placentula Ehrenberg 83.33 Cocconeis placentula var. klinoraphis Geitler 66.67 Cocconeis pseudolineata (Geitler) Lange-Bertalot 8.33 Cocconeis robusta A. Jurilj 25 Cyclotella meneghiniana Kützing 8.33 Cyclotella sp. 75 Cymatopleura apiculata W. Smith 8.33 Cymatopleura elliptica (Brébisson) W. Smith 25 Cymbella affinis Kützing 33.33 Cymbella excisa Kützing 8.33 Cymbella excisiformis Krammer 8.33 Cymbella hungarica (Grunow) Pantocsek 8.33 Cymbella hustedtii var. crassipunctata Lange-Berta- lot et Krammer 8.33 Cymbella laevis Nägeli 25 Cymbella parva (W.Smith) Kirchner 16.67 Cymbella parvula Krasske 8.33 Cymbella sp. 16.67 Cymbella subleptoceros Krammer 16.67 Denticula elegans Kützing 8.33 Denticula tenuis Kützing 33.33 Denticula tenuis var. crassula (Nägeli) Hustedt 41.67 Diatoma vulgaris Bory 91.67 Diatoma vulgaris var. capitulata Grunow 83.33 Diatoma vulgaris var. producta Grunow 8.33 Diploneis oblongella (Nägeli ex Kützing) A. Cleve 25 Ellerbeckia arenaria (Moore ex Ralfs) R.M.Crawford 66.67 Encyonema leibleinii (C. Agardh) W.J.Silva, R.Jahn, T.A.Veiga Ludwig et M.Menezes 25 Encyonema minutum (Hilse) D.G.Mann 8.33 Taxon Max (%) Encyonema silesiacum (Bleisch) D.G.Mann 66.67 Encyonema ventricosum (C. Agardh) Grunow 91.67 Encyonopsis microcephala (Grunow) Krammer 8.33 Epithemia adnata var. saxonica (Kützing) R.M.Patrick 8.33 Epithemia muelleri Fricke 25 Epithemia sp. 8.33 Epithemia turgida (Ehrenberg) Kützing 16.67 Eunotia arcus Ehrenberg 16.67 Eunotia valida Hustedt 8.33 Fragilaria vaucheriae (Kützing) J.B.Petersen 41.67 Fragilaria recapitellata Lange-Bertalot et Metzeltin 8.33 Fragilaria sp. 41.67 Gomphonema acuminatum Ehrenberg 33.33 Gomphonema angustum C. Agardh 33.33 Gomphonema apicatum Ehrenberg 8.33 Gomphonema augur Ehrenberg 41.67 Gomphonema auritum A.Braun ex Kützing 8.33 Gomphonema calcareum Cleve 25 Gomphonema capitatum Ehrenberg 8.33 Gomphonema constrictum Ehrenberg 8.33 Gomphonema gracile Ehrenberg 8.33 Gomphonema grunowii R.M.Patrick et Reimer 8.33 Gomphonema micropus Kützing 8.33 Gomphonema minutum (C.Agardh) C.Agardh 8.33 Gomphonema montanum (J. Schumann) Grunow 8.33 Gomphonema olivaceum (Hornemann) Brébisson 91.67 Gomphonema parvulum (Kützing) Kützing 16.67 Gomphonema productum (Grunow) Lange-Bertalot et Reichardt 8.33 Gomphonema pseudoaugur Lange-Bertalot 8.33 Gomphonema pumilum (Grunow) E. Reichardt et Lange-Bertalot 8.33 Gomphonema sp. 50 Gomphonema subclavatum (Grunow) Grunow 25 Gomphonema tergestinum (Grunow) Fricke 25 Gomphonema truncatum Ehrenberg 33.33 Gyrosigma acuminatum (Kützing) Rabenhorst 41.67 Gyrosigma attenuatum (Kützing) Rabenhorst 8.33 Gyrosigma sciotoense (W.S.Sullivant) Cleve 8.33 Gyrosigma sp. 16.67 Halamphora veneta (Kützing) Levkov 8.33 Hantzschia amphioxys (Ehrenberg) Grunow 25 Iconella helvetica (Brun) Ruck et Nakov 33.33 Iconella linearis (W.Smith) Ruck et Nakov 25 Iconella spiralis (Kützing) E.C.Ruck et T.Nakov 8.33 Melosira sp. 33.33 Melosira varians C.Agardh 91.67 Meridion circulare (Greville) C.Agardh 100 Navicula amphibola Cleve 8.33 Navicula capitatoradiata H.Germain ex Gasse 8.33 Navicula cari Ehrenberg 16.67 BENTHIC DIATOMS OF SMALL KARSTIC RIVER ACTA BOT. CROAT. 80 (2), 2021 163 However, similarity in species compostion was much higher between sampling periods at the first location of Bunica River (40.1%) than at the second (29%) or third (25.7%) locations. The most abundant taxa at Bunica 1 were: E. ventricosum, E. silesiacum and Planothidium lanceolatum; at Bunica 2 Halamphora veneta, C. euglypta and C. placentula and at Bunica 3 G. olivaceum, Achnanthes sp. and C. placentula var. klinoraphis. Hierarchical group average clustering and the SIMPROF test followed by SIMPER analysis identified two groups and three ungrouped samples. Figure 3 shows the results of PCO analysis conducted using a Bray Curis simi- larity matrix and overlain with hierarchical clustering with Taxon Max (%) Navicula cincta (Ehrenberg) Ralfs 16.67 Navicula cryptocephala Kützing 75 Navicula cryptonella Lange-Bertalot 16.67 Navicula jakovljevicii Hustedt 16.67 Navicula lanceolata Ehrenberg 8.33 Navicula microdigitoradiata Lange-Bertalot 8.33 Navicula oblonga (Kützing) Kützing 25 Navicula radiosa Kützing 50 Navicula reinhardtii (Grunow) Grunow 50 Navicula tripunctata (O.F.Müller) Bory de Saint- Vincent 91.67 Navicula trivialis Lange-Bertalot 25 Navicula veneta Kützing 8.33 Navicula viridula (Kützing) Ehenberg 8.33 Navicymbula pusilla (Grunow) K.Krammer 8.33 Navigeia decussis (Østrup) Bukhtiyarova 8.33 Neidium dubium (Ehenberg) Cleve 8.33 Nitzschia acicularis (Kützing) W.Smith 16.67 Nitzschia capitellata Hustedt 8.33 Nitzschia dissipata (Kützing) Rabenhorst 8.33 Nitzschia linearis W. Smith 41.67 Nitzschia palea (Kützing) W.Smith 16.67 Nitzschia pusilla Grunow 8.33 Nitzschia recta Hantzsch ex Rabenhorst 8.33 Nitzschia sigmoidea (Nitzsch) W.Smith 33.33 Nitzschia sp. 41.67 Nitzschia sublinearis Hustedt 41.67 Odontidium mesodon (Kützing) Kützing 33.33 Pinnularia microstauron (Ehrenberg) Cleve 8.33 Pinnularia sudetica Hilse 16.67 Pinnularia sp. 8.33 Placoneis gastrum (Ehrenberg) Mereschkovsky 8.3 Planothidium hauckianum (Grunow) Round et Bukhtiyarova 8.33 Planothidium lanceolatum (Brébisson ex Kützing) Lange-Bertalot 25 Reimeria sinuata (W. Gregory) Kociolek et Stoermer 16.67 Rhoicosphenia abbreviata (C. Agardh) Lange-Bertalot 91.67 Sellaphora bacillum (Ehrenberg) D.G.Mann 41.67 Sellaphora gregoryana (Cleve et Grunow) Metzeltin et Lange-Bertalot 8.33 Staurosira construens Ehrenberg 8.33 Staurosira venter (Ehrenberg) Cleve et Moeller 16.67 Staurosirella lapponica (Grunow) D.M.Williams et Round 8.33 Surirella angustata Kützing 41.67 Surirella grunowii Kulikovskiy, Lange-Bertalot et Witkovski 16.67 Surirella librile (Ehrenberg) Ehrenberg 33.33 Surirella minuta Brébisson ex Kützing 8.33 Tryblionella angustata W.Smith 8.33 Ulnaria acus (Kützing) Aboal 8.33 Ulnaria danica (Kützing) Compère et Bukhtiyarova 41.67 Ulnaria oxyrhynchus (Kützing) M.Aboal 25 Ulnaria ulna (Nitzsch) Compère 100 Fig. 3. Principal coordinates analysis (PCO) diagram comparing the community composition between samples in different sea- sons. PCO plot coded by the species vectors indicates taxa (A) and environmental vectors (B) positively correlated to PC1 and PC2 axes (r > 0.5; r > 0.3, respectively), and overlapped with the cluster groups based the 35% resemblance level of the group average clus- tering. Abbreviations: ACSP – Achnanthes sp., CPED – Cocconeis pediculus, CPLL – C. lineata, CPLK – C. placentula var. klinora- phis, CPLE – C. euglypta, MVAR – Melosira varians, EVEN – En- cyonema ventricosum, ESIL – E. silesiacum, GOLI – Gomphonema olivaceum, MCIR – Meridion circulare, NRAD – Navicula radio- sa, NTRI – N. tridentula, UULN – Ulnaria ulna; T – temperature, NH4+ – ammonium, SiO2 – silica, EC –conductivity, PO43- – or- thophosphate. Tab. 3. Continued DEDIĆ A., HAFNER D., ANTUNOVIĆ A., KAMBEROVIĆ J., STANIĆ-KOŠTROMAN S., KELLY M. G. 164 ACTA BOT. CROAT. 80 (2), 2021 vectors given by the Pearson correlation coefficient with en- vironmental data and most significant taxa. The first group on the plot included all samples from location 1, except win- ter samples, summer and winter samples from location 2, and spring and winter samples from location 3. This group was characterized by G. olivaceum (GOLI), E. ventricosum (EVEN), and M. circulare (MCIR) up to 35% cumulative contribution and is associated with lower values of PO43–, consumption of chemical oxygen demand (COD) and elec- trical conductivity (EC). The second group is composed of autumn samples from the second and third location and was characterized by C. euglypta (CPLE) and C. placentula var. klinoraphis (CPLK) with up to 50% cumulative contribu- tion; these samples are associated with higher values of SiO2 and NO3–. The ungrouped samples were 1d (dominated by P. lanceolata (ALAN) and C. placentula (CPLA), 2a with high abundance of Ulnaria oxyrhynchus (UOXY) and H. veneta (AVEN) and 3b, dominated by Cymbella laevis (CLAE) and Nitzschia pusilla (NIPU). Diatom indices The data were used to calculate values of IPS and TI for all samples. IPS calculations used 95–100% of diatoms per sample whilst TI calculations used 65–95%. The values of indices for IPS ranged from 8.9 to 18.5 and for TI from 4.9 to 12.6. The two indices were not significantly correlated with each other or with measured physical and chemical parameters (P > 0.05). The pICM values ranged from 0.21 (2a) to 1 (2c) (Tab. 5), with mean values suggesting good sta- tus at Bunica 3 but not at the other two sites (based on an intercalibrated boundary value of 0.69). The concentrations of physical and chemical parameters should mean that the Bunica River can support high ecological status (Tab. 5), so the reason for this mismatch needs to be explored. Biotic index classes assigned by Omnidia showed variations in re- sults from high to low ecological status. IPS values mostly in autumn season on all sites referred to the first category, while TI values mostly varied from fourth to fifth category, Tab. 4. Values of diversity and evenness indices in 12 samples at three stations on the Bunica River. H´ – Shannon’s Diversity Index, 1-lambda – Simpson’s Diversity Index, d – Margalef ’s Diversity Index, J – Pielou’s Evenness Index. Station Season Sample code H´ 1-lambda J d BU N IC A 1 spring 1a 1.94 0.71 0.24 4.50 summer 1b 2.51 0.88 0.41 4.82 autumn 1c 2.17 0.80 0.20 6.94 winter 1d 1.88 0.69 0.21 4.82 BU N IC A 2 spring 2a 1.85 0.69 0.20 5.40 summer 2b 2.75 0.91 0.41 6.15 autumn 2c 2.72 0.88 0.29 8.42 winter 2d 2.64 0.87 0.35 6.48 BU N IC A 3 spring 3a 2.44 0.85 0.25 7.27 summer 3b 2.26 0.81 0.25 6.13 autumn 3c 2.40 0.81 0.22 8.09 winter 3d 2.11 0.77 0.20 6.45 Tab. 5. Values of phytobenthos Intercalibration Common Metric (pICM) and water quality classes according to diatom indices (IPS and TI) and chemical conditions at three stations on the Bunica River. IPS – Specific Pollution-sensitivity Index (CEMAGREF 1982), TI –Trophic Index (Rott et al. 1999), NH4+ – ammonium, NO3-– nitrate, TN – total nitrogen, TP – total phosphorus. Station Season Sample code IPS TI pICM NH4+ NO3- TN TP BU N IC A 1 spring 1a II V 0.57 I I I I summer 1b I V 0.64 I I I I autumn 1c I V 0.77 I I I I winter 1d II V 0.47 I I I I BU N IC A 2 spring 2a IV V 0.21 I I I I summer 2b II V 0.61 I I I I autumn 2c I III 1 I I I I winter 2d II V 0.61 I I I I BU N IC A 3 spring 3a I V 0.63 I I I I summer 3b II IV 0.74 I I I I autumn 3c I IV 0.76 I I I I winter 3d I V 0.70 I I I I BENTHIC DIATOMS OF SMALL KARSTIC RIVER ACTA BOT. CROAT. 80 (2), 2021 165 indicating extremely high trophic status (Tab. 5). However, these classes are over-simplistic and do not give nuanced insights into conditions in any particular river. The percent- age of pollution tolerant taxa for site 1 were 0.3% in spring, 1.8% in summer, while for autumn and winter they were not registered. For site 2 there were 39.7 pollution tolerant taxa registered and for site 3 those taxa were not recorded. Discussion In this paper the benthic diatom assemblages in karstic river in BH were studied in order to estimate ecological sta- tus and to test the future applicability of diatom indices. Benthic diatoms have been recommended in recent decades as appropriate tools for pollution assessment in rivers (e.g., Coste et al. 1991, Whitton and Kelly 1995), and have been used worldwide (Porter et al. 2008, Mangadze et al. 2015). Although our results indicate that the nutrient concentra- tions of the water do not determine the composition and structure of the benthic diatom assemblage in the Bunica River, this is likely to be due to the short gradient encoun- tered during this study. All sites in all seasons had low nu- trient concentrations and good oxygenation. The high oxy- gen saturation and water transparency, as well as low nutrient concentrations, indicate the oligotrophic character of the river. For the investigated stations in different sea- sons, no major seasonal fluctuations and differences in physical and chemical parameters were identified, which is probably due to the proximity and impact of springs along this short river (just 5.8 km in length). Water temperature (11–17 °C) showed relatively low values for all seasons. The smallest fluctuations were closest to the spring due to the temperature stability of spring water. High values of dis- solved oxygen (9.65–13.3 mg L–1) and saturation (90.1–132%) are due to lower water temperature, porosity of the rock aquifer and contact between groundwater and the atmo- sphere (Cantonati 1998). Higher conductivity values indi- cate higher mineralization and our results suggest that the Bunica River has moderate mineralization. Physical and chemical values in this study are in accordance with values for limestone substrate (Mogna et al. 2015). The analysis of the diatom assemblages revealed high species diversity. The investigated sites in the Bunica River were characterized by similar numbers of diatom taxa, and high species diversity indicates that there were favorable conditions for development. A similar number of diatom taxa (117) was recorded in the Mura River (Krivograd- Klemenčić and Balabanič 2010), while in the Bunica spring 104 (Dedić et al. 2015) and 87 (Hafner and Jasprica 2010) taxa were recorded. Although this is not universally appli- cable, similar correspondence between low levels of pollu- tion and high species diversity were also observed by Noga et al. (2014). The Bunica River has a low level of organic pol- lution and it is not impacted by many anthropogenic stress- ors (high density of population, livestock farming, devel- oped industry, etc.). This was also supported by diversity and evenness indices. Although values of indices varied for longitudinal and temporal patterns they indicated that the structure of habitats were stabile and balanced. Their varia- tions reflect the heterogeneity of the diatom population. Those genera that were well represented in this study (Gom- phonema, Navicula and Nitzchia) were also well represented in other studies of karstic rivers (Wojtal 2009, Dedić et al. 2015). All three genera are found across the whole range of the Bunica River, with particular species favouring different levels of ions and nutrients. Meridion circulare and Ulnaria ulna were recorded in all samples. Meridion circulare is a widespread, mesosaprobous and oligo to eutraphentic alka- liphilous diatom (Van Dam et al. 1994). Ulnaria ulna occurs in oligo- to polytrophic and oligo-saprobic to α-mesosaprobic waters (Hofmann et al. 2013) and is also classified as alka- liphilous (Van Dam et al. 1994). The occurence of large numbers Halamphora veneta in a spring at the middle loca- tion is, however, very unusual. It occurs in fresh and slight- ly brackish water. According to Van Dam et al. (1994) it is an alpha/polisaprobic and eutrophic taxa. Taxa of the genera Amphora, Cocconeis and Diatoma, frequently found in this research, have a high indicator val- ues for pH, organic nitrogen, oxygen, saprobity and trophic state (Van Dam et al. 1994). Diatoms are influenced by ma- ny factors including those that are site-specific on various temporal and species scales (DeNicola et al. 2004) as well as those that reflect human interventions in the environment. Those factors include chemical properties of the water (Po- tapova 1996), nutrient load (Rott et al. 1997) and also flow velocity and substratum type (Rimet 2009). The results of this study showed that physical and chemical variables and seasons did not have a notable effect on the diversity of ben- thic diatoms in the Bunica River. It is possible that hydro- morphological and other water conditions such as flow ve- locity, type of substrates, discharge of water, marginal vegetation in karstic river also influenced the structure and composition of benthic diatom assemblages more than physical and chemical variables. IPS and TI did not always give consistent results along the entire length of the Bunica River. One important reason is that they were developed for different purposes – IPS measures “general degradation” whilst TI was developed, specifically, to measure the impact of inorganic nutrients. IPS index showed better water quality along the entire length of the Bunica river (mostly moderate, II and III class) compared with the TI trophic index, which indicated a poor or bad ecological status of the river (IV-V class). This vari- ation has also been noted by others, Kitner and Poulícková (2003), Poulíčková et al. (2004), and Stenger-Kovács et al. (2007), Szczepocka and Szulc (2009), Kalyoncu and Şerbetci (2013). Because the indices were developed for different purposes, naive division of the scale into equal-sized seg- ments, though an easy option (e.g., Eloranta and Soininen 2002), is unlikely to give meaningful results or consistency between indices. Others have noted that indices developed in certain regions of Europe were not effective in others (Pipp 2002, Rott et al. 2003). DEDIĆ A., HAFNER D., ANTUNOVIĆ A., KAMBEROVIĆ J., STANIĆ-KOŠTROMAN S., KELLY M. G. 166 ACTA BOT. CROAT. 80 (2), 2021 Our study also noted a mismatch between status assess- ment using the average pICM boundary set during the Med- iterranean intercalibration exercise (Almeida et al. 2014) and observations in this study. The nature of the Bunica River is such that we should estimate high or good status based on diatom assemblages, whereas our results indicate less than good status at two of the sites. Kamberović et al. (2019 a) used TI index in a study of springs on Konjuh Mountain (BH) and reached similar conclusions about the suitability of existing indices, recommending some modi- fications to the TI. Our study has also identified potential mismatches between ecological status boundaries used else- where in the Mediterranean basin and those used in BH, suggesting a need to recalibrate the pICM, if it is to be used successfully here. Based on this study, we recommend “ex- pected” values for pICM calculations of 16.8 and 7.3 for IPS and TI, respectively. Diatom indices have been shown to be one of the most effective tools for evaluating ecological status in European rivers (Eloranta and Soininen 2002, Kelly et al. 2008); how- ever, further testing of IPS and TI diatom indices was need- ed to ensure their utility in small karstic rivers in BH. The suitability of indices depends, to some extent, on the per- centage of taxa used for index calculation. In this study 95– 100% of taxa were used for the IPS calculation, suggesting that it is highly suitable. For the TI, a smaller percentage was used (60–90%). In the study of epiphytic diatoms in Lake Modrac in northeastern BH, Kamberović et al. (2019b) also concluded that the IPS and TI were most suitable. However, consideration should also be given to how sensitivity values are assigned to taxa and, in this respect, the IPS does not have a transparent process for determining such scores whilst the TI has never been updated since first published (Rott et al. 1999). On the other hand, both are widely used around Europe (Poikane et al. 2016) and, therefore provide a means by which results from BH can be compared with data from other countries. Rather than develop a new index, therefore, we have adopted the ‘‘phytobenthos Intercalibra- tion Common Metric’’ (pICM, Kelly et al. 2009), based on these two metrics, as an interim tool for evaluating ecolog- ical status using phytobenthos in BH. These values are sim- ilar to those proposed for Mediterranean streams by Kelly et al. (2012), and higher similarities were recorded in IPS than in TI, but have the benefit for being more closely tuned to local conditions within BH. Conclusions The Bunica River has a high diversity of benthic diatom taxa (147). Physical and chemical analyses indicated oligo- trophic conditions in all locations and high ecological sta- tus. Measured physical and chemical parameters and sea- sons did not have a notable effect on the biological diversity of benthic diatoms. It is possible that hydromorphological and other water conditions such as flow velocity, type of substrates, discharge of water, marginal vegetation had a greater influence on structure and composition of benthic diatom taxa than physical and chemical variables. The diatom indices IPS and TI were used in our study and we concluded that combining them into the “phytobenthos Intercalibration Common Metric’’ (pICM) gave us more results that were more easily compared with those from elsewhere in Europe than would have been the case if only a single index had been used. We therefore recommend pICM as a good starting point for ecological status assess- ment in BH and propose it as a potential national metric for phytobenthos assessment in the future. However, testing on a larger number of sites and along a longer gradient of eco- logical quality is necessary before this can be confirmed. References Almeida, S.F.P., Elias, C., Ferreira, J., Tornés, E., Puccinelli, C., Delmas, F., Dőrflinger, G., Urbanič, G., Marcheggiani, S., Rosebery, J., Mancini, L., Sabater, S., 2013: Water quality as- sessment of river using diatom metrics across Mediterranean Europe: A methods intercalibration exercise. Science of the Total Environmental 476–477, 768–776. Anonymous, 2014: Decision on surface and groundwater char- acterization, reference conditions and parameters for water status assessment and water monitoring. Official Gazzete of Federation of Bosnia and Herzegovina 01/14, 61–119 (in Bos- nian/Croatian/Serbian). Cantonati, M., 1998: Diatom communities of springs in the Southern Alps. Diatom Research 13, 201–220. CEMAGREF, 1982: Etude des méthods biologiques quantitatives d’appréciation de la qualité des eaux. Rapport Division Qual- ite des Eaux Lyon – Agence financière de Bassin Rhône – Méditerranée – Corse, Pierre-Bénite. CEN, 2003: The European Standard. Water quality. Guidance standard for the routine sampling and pre-treatment of ben- thic diatoms from rivers. EN 13946. European Committee for Standardization, Brussels. Clarke, K.R., Gorley, R.N., 2006: Primer v.6. PRIMER-e, Plym- outh. Coste, M., Boca, C., Dauta, A., 1991: Use of algae for monitoring rivers in France. In: Whitton, B.A., Rott, E., Friedrich, G. (eds.), Use of algae for monitoring rivers, 75–88. Institut für Botanik, Universität Innsbruck, Innsbruck, Austria. Dedić, A., 2015: Dynamics of periphytic diatom colonization in karst springs of Bosnia and Herzegovina. PhD Thesis. Fac- ulty of Science, University of Zagreb, Zagreb (in Croatian). Dedić, A., Plenković-Moraj, A., Borojević Kralj, K., Hafner, D., 2015: The first report on periphytic diatoms on artificial and natural substrate in the karstic spring Bunica, Bosnia and Herzegovina. Acta Botanica Croatica 74, 393–406. DeNicola, D.M., Eyto, E.D., Wemaere, A., Irvine, K., 2004: Us- ing epilithic algal communities to assess trophic staus in Irish lakes. Journal of Phycology 40, 481–495. Directive, 2000: Directive 2000/60/EC of the European Parlia- ment and the council of 23 October 2000 establishing a framework for community action in the field of water policy. Official Journal of the European Communities L 327, 1–72. Eloranta, P., Soininen, J., 2002: Ecological status of some Finn- ish rivers evaluated using benthic diatom communities. Journal of Applied Phycology 14, 1–7. Galić, A., 2011: Hydrogeological conditions of the area of water reservoirs in western Herzegovina. PhD Thesis. Faculty of Mining, Geology and Civil Engineering, University of Tu- zla, Tuzla (in Bosnian). BENTHIC DIATOMS OF SMALL KARSTIC RIVER ACTA BOT. CROAT. 80 (2), 2021 167 Guiry, M.D., Guiry, G.M., 2020: AlgaeBase. World-wide elec- tronic publication, National University of Ireland, Galway. Retrieved January 14, 2020 from http://www.algaebase.org Hafner, D., Jasprica, N., 2010: Taxonomic composition and sea- sonality of diatoms in three karstic rivers in Herzegovina (Bosnia and Herzegovina). In: Kusber, W., Jahn, R. (eds.), Proceedings of the Abstracts of the 4th Central European Diatom Meeting, 24–25, Reichenau, Bodensee. Hammer, Ø., Harper, D.A.T., Ryan, P., 2001: PAST. Paleonto- logical statistics software package for education and data analysis. Palaeontologia Electronica 4, 9. Hofmann, G., Werum, M., Lange-Bertalot, H., 2013: Diatomeen im Süßwasser-Benthos von Mitteleuropa. Bestimmungsflo- ra Kieselalgen für die ökologische Praxis. Koeltz Scientific Books, Königstein. Kalyoncu, H., Şerbetci, B., 2013: Applicability of Diatom-Based Water Quality Assessment Indices in Dari Stream, Isparta/ Turkey. International Journal of Environmental, Chemical, Ecological, Geological and Geophysical Engineering, 7, 386– 394. Kamberović, J., Plenković-Moraj, A., Kralj Borojević, K., Gligo- ra Udovič, M., Žutinić, P., Hafner, D., Cantonati, M., 2019a: Algal assemblages in springs of different lithology (ophiolites vs. limestone) of the Konjuh Mountain (Bosnia and Herze- govina). Acta Botanica Croatica 78, 66–81. Kamberović, J., Stuhli, V., Lukić, Z., Habibović, M., Mešikić, E., 2019b: Epiphytic diatoms as bioindicators of trophic status of Lake Modrac (Bosnia and Herzegovina). Turkish Journal of Botany 43, 420–430. Kelly, M., Juggins, S., Guthrie, R., Pritchard, S., Jamieson, J., Rippey, B., Yallop, M., 2008: Assessment of ecological status in UK rivers using diatoms. Freshwater Biology 53, 403–422. Kelly, M., Bennett, C., Coste, M., Delgado, C., Delmas, F., Denys, L., Ector, L., Fauville, C., Ferréol, M., Golub, M., Jarlman, A., 2009: A comparison of national approaches to setting eco- logical status boundaries in phytobenthos assessment for the European Water Framework Directive: results of an inter- calibration exercise. Hydrobiologia 621, 169–182. Kelly, M.G., Gómez-Rodríguez, C., Kahlert, M., Almeida,S.F.P., Bennett, C., Bottin, M., Delmas, F., Descy, J-P., Dőrflinger, G., Kennedy, B., Marvan, P., Opatrilova, L., Pardo, I., Pfister, P., Rosebery, J., Schneider, S., Vilbaste, S., 2012: Establishing expectations for pan-European diatom based ecological sta- tus assessments. Ecological Indicators 20, 177–186. Kitner, M., Poulícková, A., 2003: Littoral diatoms as indicators for the eutrophication of shallow lakes. Hydrobiologia 506, 519–524. Krammer, K., 2000–2003: The Genus Pinnularia, 1, 703; Cym- bella, 3, 584; Cymbopleura, Delicata, Navicymbula, Gompho- cymbellopsis, Afrocymbella, 4, 530 In: Lange-Bertalot, H. (ed.), Diatoms of Europe. TA.R.G. Gantner Verlag K.G, Rug- gell, Liechtenstein. Krammer, K., Lange-Bertalot, H., 1986: Bacillariophyceae, 1. Teil: Naviculaceae. In: Ettl, H., Gerloff, J., Heynig, H., Mol- lenhauer, D. (eds.), Süsswasserflora von Mitteleuropa 2/1. G. Fischer-Verlag, Stuttgart. Krammer, K., Lange-Bertalot, H., 1988: Bacillariophyceae, 2. Teil: Bacillariaceae, Epithemiaceae, Surirellaceae. In: Ettl, H., Gerloff, J., Heynig, H., Mollenhauer, D. (eds), Süsswasser- flora von Mitteleuropa 2/2. G. FischerVerlag, Stuttgart. Krammer, K., Lange-Bertalot, H., 1991: Bacillariophyceae, 3. Teil: Centrales, Fragilariaceae, Eunotiaceae. In: Ettl, H., Ger- loff, J., Heynig, H., Mollenhauer, D. (eds.), Süsswasserflora von Mitteleuropa 2/3. G. Fischer-Verlag, Stuttgart. Krammer, K., Lange–Bertalot, H., 2004: Bacillariophyceae, Ach- nanthaceae, 2/4. In: Ettl, H., Gärtner, G., Heynig, H., Mol- lenhauer, D. (eds.), Sűβwasserflora von Mitteleuropa. G. Fischer, Stuttgart, New York. Krivograd-Klemenčič, A., Balabanič, D., 2010: Phytobenthos and water quality in the Mura River’s oxbows. Natura Slo- veniae 12(2), 5–22. Lange-Bertalot, H., 2001: Navicula sensu stricto, 10 genera sep- arated from Navicula sensu lato, Frustulia. In: Lange-Berta- lot, H. (ed.), Diatoms of Europe, 2. A.R.G. Gantner Verlag K.G., Ruggell. Lecointe, C., Coste, M., Prygiel, J., 1993: “Omnidia”: software for taxonomy, calculation of diatom indices and inventories management. Hydrobiologia 269/270, 509–513. Mangadze, T., Bere, T., Mwedzi, T., 2015: Epilithic diatom flora in contrasting land-use settings in tropical streams, Man- yame Catchment, Zimbabwe. Hydrobiologia 753, 163–173. Milanović, P.T., 2006: Karst of eastern Herzegovina and Du- brovnik littoral. ASOS, Belgrade (in Serbian). Mogna, M., Cantonati, M., Andreucci, F., Angeli, N., Berta, G., Miserere, L., 2015: Diatom communities and vegetation of springs in the south-western Alps. Acta Botanica Croatica 74, 265–285. Mrđen, D., Matković, I., Šarac, M., 2018: Preparation of water management plan for Adriatic river basin district in Federa- tion of B&H. e-Zbonik: Electronic Collection of Papers of the Faculty of Civil Engineering 8(15), 77–85. Noga, T., Stanek-Tarkowska, J., Pajączek, A., Kochman, N., Peszek, Ł., 2014: Ecological assessment of the San River wa- ter quality on the area of the San valley Landscape park. Journal of Ecological Engineering 15, 12–22. Pardo, I., Delgado, C., Abraín, R., Gómez-Rodríguez, C., García- Roselló, E., García, L., Reynoldson, T.B., 2018: A predictive diatom-based model to assess the ecological status of streams and rivers of Northern Spain. Ecological Indicators 90, 519– 528. Pardo, I., Gómez-Rodríguez, C., Wasson, J. G., Owen, R., van de Bund, W., Kelly, M., Mengin, N., 2012: The European refer- ence condition concept: a scientific and technical approach to identify minimally-impacted river ecosystems. Science of the Total Environment 420, 33–42. Pardo, L.H., Fenn, M.E., Goodale, C.L., Geiser, L.H., Driscoll, C.T., Allen, E.B., Emmett, B., 2011: Effects of nitrogen depo- sition and empirical nitrogen critical loads for ecoregions of the United States. Ecological Applications 21, 3049–3082. Pipp, E., 2002: A regional diatom-based trophic state indication system for running water sites in Upper Austria and its over- regional applicability. Verhandlungen des Internationalen Verein Limnologie 27, 3376–3380. Poikane, S., Kelly, M.G., Cantonati, M., 2016: Benthic algal as- sessment of ecological status in European lakes and rivers: challenges and opportunities. Science of the Total Environ- ment 568, 603–613. Porter, S.D., Mueller, D.K., Spahr, N.E., Munn, M.D., Dubrovsky, N.M., 2008: Efficacy of algal metrics for assessing nutrient and organic enrichment in flowing waters. Freshwater Biol- ogy 53(5), 1036–1054. Potapova, M., 1996: Epilithic algal communities in rivers of the Kolyma mountains, NE Siberia, Russia. Nova Hedwigia 63, 309–334. Poulíčková, A., Duchoslav, M., Dokulil, M., 2004: Littoral dia- tom assemblages as bioindicators of lake trophic status: A case study from perialpine lakes in Austria. European Jour- nal of Phycology 39, 143–152. Rimet, F., 2009: Benthic diatom assemblages and their corre- spondence with ecoregional classifications: case study of riv- ers in north-eastern France. Hydrobiologia 636, 137–151. DEDIĆ A., HAFNER D., ANTUNOVIĆ A., KAMBEROVIĆ J., STANIĆ-KOŠTROMAN S., KELLY M. G. 168 ACTA BOT. CROAT. 80 (2), 2021 Rott, E., Hofmann, G., Pall, K., Pfister, P., Pipp E., 1997: Indika- tionslisten für Aufwuchsalgen. Teil 1: Saprobielle Indikation. Bundesministerium für Land- und Forstwirtschaft, Wien. Rott, E., Pfister, P., Van Dam, H., Pipp, E., Pall, K., Binder, N., Ortler, K., 1999: Indikationslisten fur Aufwuchsalgen. Teil 2: Trophieindikation sowie geochemische Pra ¨ferenz, tax- onomische und toxikologische Anmerkungen. Bundesmin- isterium für Land- und Forstwirtschaft, Wien. Rott, E., Pipp, E., Pfister, P., 2003: Diatom methods developed for river quality assessment in Austria and a cross-check against numerical trophic indication methods used in Eu- rope. Algological Studies 110, 91–115. Stenger-Kovács, C., Buczko, K., Hajnal, E., Padisák, J., 2007: Ep- iphytic, littoral diatoms as bioindicators of shallow lake tro- phic status: Trophic Diatom Index for Lakes (TDIL) devel- oped in Hungary. Hydrobiologia 589, 141–154. Szczepocka, E., Szulc, B., 2009: The use of benthic diatoms in estimating water quality of variously polluted rivers. Ocean- ological and Hydrobiological Studies 38, 17–26. Van Dam, H., Mertens, A., Sinkeldam, J., 1994: A coded check- list and ecological indicator values of freshwater diatoms from the Netherlands. Netherland Journal of Aquatic Ecol- ogy 28, 117–133. Van de Bund, W.J., 2009: Water Framework Directive Intercali- bration Technical Report. Part 1: Rivers. JRC Scientific and Technical Reports 179. Whitton, B.A., Kelly, M., 1995: The trophic diatom index: a new index for monitoring eutrophication in rivers. Journal of Ap- plied Phycology 7, 43–444. Wojtal, A.Z., 2009: The diatoms of Kobylanka Stream near Kraków (Wyżyna Krakowsko-Częstochowska Upland, S Po- land). Polish Botanical Journal 54, 129–330.