ECOLOGICAL STATUS ASSESSMENT OF THE SAMOKOVSKA RIVER-2018 CASE STUDY

The Samokovska River, as the most important watercourse of the Kopaonik National Park, has been poorly investigated from algological and water quality aspects. This river is not covered by the monitoring program of the Serbian Environmental Protection Agency routine. Our research performed in October 2018 aimed to present an algal diversity, ecological status assessment, and negative anthropogenic impacts threatening this river. For ecological status assessment purposes, the diatom indices phytobenthos and physico-chemical parameters of water were used. The typology and the problem of reliable assessing of the ecological status were also discussed on the example of the Samokovska River.


INTRODUCTION
Due to the Law on National Park Kopaonik dating from 1981 (ANONYMOUS, 1981), this mountain was declared to be a National Park and was put under the country's protection as global common goods. The Samokovska River is the main watercourse on Kopaonik. It covers the area of 66.66 ha. As a part of the National Park, the Samokovska River has a privileged status of being a nature reserve (VASOVIĆ, 1988).
The Samokovska River was poorly investigated from algological and water quality aspects. The first, as well the only literature data on the algological and saprobiological research of this river date from the 1990s (LAUŠEVIĆ 1992(LAUŠEVIĆ , 1993LAUŠEVIĆ and CVIJAN 1996). Thereafter, presence of some benthic macroalgae in the Samokovska River was reported by SIMIĆ (2002), SIMIĆ et al. (2003), and SIMIĆ and ĐORĐEVIĆ (2017).
According to the National Regulations (ANONYMOUS, 2010b, 2011b), all Serbian surface waters are classified into six types according to the criteria for water bodies typology such as geology, basin surface area, altitude, and type of substrate as the auxiliary parameter: Type 1 -large lowland rivers with domination of fine substrate; Type 2 -large rivers with domination of medium sized substrate, excluding rivers of Pannonian Basin; Type 3 -small and medium rivers, up to 500 m altitude and domination of large substrate granulation; Type 4 -small and medium rivers at altitude above 500 m and domination of large granulated substrate; Type 5 -Pannonian Basin rivers, excluding rivers from Type 1; Type 6 -small watercourses outside the Pannonian Basin area not being included in Types 3 and 4, as well as the watercourses not encompassed by the National Regulation (ANONYMOUS, 2010b). Since the Samokovska River is not covered by the National Regulation (ANONYMOUS, 2010b), it belongs to the water bodies of Type 6. However, according to the previously mentioned criteria for water bodies' typology, the Samokovska River can be placed to the water body Type 4.
For ecological status assessment of surface water, use of phytobenthos community parameters is recommended by Water Framework Directive (WFD, 2000). Although the term phytobenthos relates to all algae inhabiting the bottom of aquatic ecosystems, the National Regulation (ANONYMOUS, 2011b) comprises parameters of ecological status assessment based on IPS -"Indice de pollution-sensibilite" (CEMAGREF, 1982) and CEE -"Comission for Economical Community metric" (DESCY and COSTE, 1991) diatom indices. Diatoms are widely recognized as bioindicators of water quality because of their worldwide distribution, ability to inhabit various habitats, to be present in aquatic ecosystems throughout the year, and very quick responses to changes in environmental conditions (MCCORMIC and CAIRNS, 1994;POIKANE et al., 2016).
This paper aimed to present an algological diversity and ecological status assessment of the Samokovska River, as well as to indicate the problematic typology of water bodies on the example of this river.

MATERIALS AND METHODS
The field research on the Samokovska River were performed on the 31 st of October 2018 on five localities.

Description of localities
Locality S1 ( Figure 1A) on the Samokovska River is positioned at the excursion site Kadijevac (N 43°19'29.1'', E 20°45'67.3'', elevation 1421 m). The locality is placed on granitic geological substratum whereas the neighbouring vegatation consists of spruce forests and peat bogs. The locality is situated in the second degree protection zone (ANONYMOUS, 2016).
Locality S2 ( Figure 1B) on the Samokovska River lies on the water intake of derivative small hydropower plant (HPP) "Samokovska reka 1" ( Figure 1C) (N 43°19'29.3'', E 20°45'47.6'', elevation 1411 m). The locality is situated on granitic geological substratum and there is a sparse spruce forest around the water intake of the small HEPP. The small HPP "Samokovska reka 1" was built in 2017 with a water intake set in the riverbed of the Samokovska River. The underground pipes start from the water intake of the HPP and they mostly pass through the protected zone of the National Park Kopaonik. The water is conducted through the pipes to the plant room placed at the entrance of the National park from the direction of Jošanička Spa. Locality S2 is positioned in the second degree protection zone (ANONYMOUS, 2016).

Sample collection
The samples of benthic algae on S1, S3, S4, and S5 were collected dependent on the types of morphological forms and substratum (by tweezers, scraping from rocks, by pipettes on sand surfaces). The samples of epilithic diatoms for ecological status estimation were collected according to standard EN 13946, 2015. The phytoplankton sample for qualitative analysis at the S2 locality was collected by plankton net (Ø 22 µm). The collected algal material was fixed in 4% formaldehyde solutuon and it was stored at the Department of Biology and Ecology of the Faculty of Science, University of Kragujevac.
The analysis of the collected algal material was performed under the light microscope Мotic BA310 at 4х, 10х, 40х and 100х magnification (oil immersion). The microphotographs of found taxa were taken by digital camera BRESSER (9МР) and by using the software package MicroCamLab. Succeeding the measurement of morphological parameters, the identification of recorded taxa was performed according to ANAGNOSTIDIS (1999, 2005)

Measurements of physical and chemical parameters of water
Physical and chemical parameters of the water were measured according to the standard EN 5667 1-19, 2017. In determining physical characteristics of the water, at each locality the direct measurements of temperature (°C) and conductivity (μs/cm 3 ) were done, whereas the chemical properties measured directly on the field included the data on dissolved oxygen concentration (mg/l), oxigen saturation (%), pH and hardness (mg/l). By using colorimeter, the concentrations of phosphate (mg/l), nitrate (mg/l), and ammonia (mg/l) were determined in the Laboratory of the Center for Fishery and Biodiversity Conservation of Inland Waters -Aquarium, Faculty of Science, University of Kragujevac.

Ecological status assessment
The ecological status of the Samokovska River was assessed using epilithic diatoms (IPS and CEE diatom indices) and supporting physico-chemical quality elements, both for water bodies belonging to Types 6 and 4. According to the National Regulation (ANONY-MOUS, 2011b), for Type 4, calculation of two diatom indices -IPS and CEE is obligatory, in contrast to the Type 6, for which only IPS index is required. Threshold values of these diatom indices between classes for all waterbody types are prescribed in the National Regulation (Table 1) (ANONYMOUS, 2011b).

Threatening factors
Possible threat factors were determined at each locality by visual analysis.

Analysis of physical and chemical parameters of water
Physical and chemical parameters of water at investigated localities of the Samokovska River indicate that water was cold, low-mineralized, soft, and well-aerated (Table 2). At the localities at higher altitudes, located in coniferous forests, pH was slightly acid, while at lower altitudes, it was slightly alkaline ( Table 2). Nutrient enrichment was low, except regarding phosphates at S3, S4, and S5 localities, which concentrations were increased ( Table  2).

Algal diversity
During researching in autumn 2018, a total of 79 taxa belonging to 5 phyla were recorded in the Samokovska River: Cyanobacteria -10, Rhodophyta -1, Ochrophyta (Xanthophyceae) -1, Bacillariophyta -63, and Chlorophyta -4. The highest species richness was recorded at S1 (55 taxa), while the lowest was recorded at S2 locality (12 taxa) ( Table 3). Only a few diatom representatives were recorded in the phytoplankton sample (S2) ( Table 3), where the phytoplankton community has not yet been formed.  Diatoms were the most common component of algal flora at all localities (Table 3). From the total number of Bacillariophyta, the largest (42 taxa) was recorded at S1, while the smallest was recorded at S2 locality (12 taxa). The highest number of taxa belonged to the genera Navicula (7), Nitzschia (7), and Achnanthidium (6).
In compared to results published by LAUŠEVIĆ (1993), during our research in autumn 2018, a smaller number of taxa were recorded in the Samokovska River, which is primarily a consequence of taking a smaller number of samples from a smaller number of localities, as well as one-time research. Previously reported finding of Batrachospermum sp. in the upper stream of the Samokovska River (SIMIĆ and ĐORĐEVIĆ, 2017) was not confirmed by our research, as well as the finding of Hydrurus foetidus (SIMIĆ et al., 2003). During our research, at all investigated localities, except S2, it has been confirmed the presence of red alga Audouinella chalybea that formed microscopic aggregations at S1 locality only. Taxonomy of this alga has often been a subject of consideration by algologists ZUCCHI, 1995, 1997;NECCHI et al., 1993aNECCHI et al., , 1993bZUCCHI and NECCHI, 2003). According to ZUCCHI and NECCHI (2003), the reddish thalli of the alga represent the Audouinella genus while the bluegreen thalli represent a "Chantransia" stage in Batrachospermales and Thoreales development.

Ecological status assessment
Based on analysis of physico-chemical parameters, prescribed by the National Regulation (ANONYMOUS, 2011b) ecological status of the Samokovska River for surface water of Type 6 could be assessed as high (class I) at S1 and as good (class II) at S3, S4 and S5 localities (Tables 2, 4).
Based on analysis of parameters of phytobenthos as a biological quality element, the ecological status of the Samokovska River could be assessed as high (class I) at S3 (IPS=16.1) and S4 (IPS=14.9) localities and as good (class II) at S1 (IPS=13.8) and S5 (IPS=13.3) localities ( Table 4).
The ultimate ecological status assessment based on biological and supporting physicochemical quality elements indicate that at all investigated localities the ecological status was good (class II) ( Table 4). Based on analysis of physico-chemical parameters, prescribed by the National Regulation (ANONYMOUS, 2011b) ecological status of the Samokovska River for water body Type 4 could be assessed as high (class I) at S1, as good (class II) at S4 and S5, and as moderate (class III) at S3 locality (Tables 2, 4).
The ultimate ecological status assessment based on biological and supporting physicochemical quality elements indicate that ecological status was good (class II) at S4 and moderate (class III) at S1, S3 and S5 localities (Table 5).  The level of reliability of ecological status assessment of the Samokovska River could be labeled as low, since for ecological assessment have not been used all relevant parameters of recommended quality elements and because the frequency of monitoring is lower than the minimum predicted.
According to the WFD (WFD, 2000), non-diatom benthic algae, including macroalgae, are recommended as useful biological quality elements, but the methodology of their usage in ecological status assessment has not been established in Serbia so far. However, their indication of water saprobity could correspond to classes of ecological status. Thus, red alga Audouinella chalybea is an indicator of oligosaprobic water, with low taxon indicator weight (SLÁDEČEK, 1973;PÁL, 1998). In Serbia, the species was found in oligosaprobic, but also in ß-mesosaprobic waters (CVIJAN and BLAŽENČIĆ, 1986;CVIJAN, 2002;CVIJAN et al., 2003;SIMIĆ et al., 2016). Cyanobacteria Microcoleus favosus and green alga Cladophora glomerata are an indicator of ß-mesosaprobic water, whereby M. favosum possess a high and C. glomerata a low taxon indicator weight (SLÁDEČEK, 1973;PÁL, 1998). According to SLÁ-DEČEK (1973) and PÁL (1998), green alga Microspora amoena is characterized as an indicator of xenosaprobic or oligosaprobic water, but its higher value of saprobic valence is in ßmesosaprobic degree and possesses a low taxon indicator weight. Green alga Ulothrix zonata is presented by two types from which one represents an indicator of oligosaprobic, and the second as an indicator of α-mesosaprobic water, both with a high taxon indicator weight (SLÁDEČEK, 1973;PÁL, 1998).
In comparison to the previous assessment of the degree of water saprobity (LAUŠEVIĆ, 1992), our results indicate deterioration of water quality of the Samokovska River. According to the water quality assessment based on macroalgae, the Samokovska River better corresponds to water body Type 4. Negative anthropogenic influences noticed in the field also support our inference.

Treatening factors
This river is threatened by various anthropogenic influences, such as an inadequately regulated sewage network in the area of the National Park Kopaonik, so a large number of sewers drains outflow into the Samokovska River and its tributaries. Results of physicochemical and biological quality elements do not indicate the water pollution since our research was conducted in October when the accommodation capacities within the tourist complex are unfilled, so the pressure of sewage was not prominent. This confirmed that onetime research results give us only a current picture of environmental conditions. However, disappearance of stenovalent red alga Batrachospermum sp. (SIMIĆ and ĐORĐEVIĆ, 2017) and golden alga Hydrurus foetidus (SIMIĆ et al., 2003) from the Samokovska River is not unexpected. The habitat of these algae has changed significantly in recent years. Intensive deforestation, pollution, and turbidity of water have conditioned the disappearance of these rare algae, creating conditions for more intensive development of competitive filamentous algae.
A small HPP "Samokovska reka 1" (Fig. 1C), located in the zone of II and III protection degrees can be singled out as an important threatening factor. Small HPPs are a negative anthropogenic factor known to cause immeasurable environmental damages on lotic ecosystems that may lead to long-term changes in the benthic algal communities, especially regarding stenovalent species (WU et al. Since the Samokovska River is the most important watercourse in the National Park Kopaonik, for trustworthy assessment of water quality of the Samokovska River, at least three times per year monitoring is necessary, but also use of all predicted parameters of biological and physico-chemical quality elements. The untrustworthy typology problem of this river is also necessary to be solved for further research.