INDICATIVE ECOLOGICAL STATUS ASSESSMENT OF THE DESPOTOVICA RIVER

The investigation of the Despotovica River was carr ied out in April 2017 with the aim to provide an indicative ecological status asse s ment. Phytobenthos and aquatic macroinvertebrate communities were followed as well as physico-chemical parameters of water. Ecological status assessment is given based on biol ogica (epilithic diatoms and macroinvertebrates) and supporting physico-chemical water quality elements, according to the Serbian National regulation on parameters of ecolog ical and chemical status of surface waters and parameters of chemical and quantitative status of ground waters. The ecological status was determined by the worst evaluated water quality ele m nt. Based on the analysis of investigated parameters of biological and physico-chemical water quality elements, the indicative ecological status of the Despotovica River could be assessed a bad (class V), which is the result of the river's load of organic and inorganic origin but al so of heavy metals.


INTRODUCTION
The investigation in the field of the traditionally used term "water quality", contributed to the establishment of a new approach to water monitoring, in which the ecological status of the aquatic ecosystem as an expression of the quality of the structure and functioning, specific for each type of water body (WFD, 2000).The Water Framework Directive regulates the management of aquatic ecosystems in the territory of the European Union and provides a new approach to water monitoring.According to the principles of the WFD (WFD, 2000), the status of surface waters assesses on the base of ecological and chemical status, whereby it has been determined by worse than ecological and chemical status (DENIĆ et al., 2015a).
According to the WFD (WFD, 2000) systemic monitoring is necessary, which includes all relevant parameters of biological quality elements, as well as parameters of supporting chemical, physico-chemical and hydromorphological quality elements.Ecological status assessment determines the worst evaluated biological quality element, while the condition of physico-chemical and hydromorphological quality elements has an indirect affect to assessment through their influence on the condition of biological quality elements and had to be aligned with evaluation of biological quality elements (DENIĆ et al., 2015a).
The implementation of the WFD (2000) in Serbia began with adopting the Water Law (ANONYMOUS, 2010a) and by-laws harmonized with them.Adopting the Serbian National regulative (ANONYMOUS, 2010b(ANONYMOUS, , 2011a;;2011b, 2012) ecological status assessment based on biological (phytoplankton, phytobenthos, macrophytes and fish) and chemical, physicochemical and hydromorphological quality elements, of relevance to biological quality elements, was established.
Monitoring of surface waters, according to WFD (WFD, 2000), and in accordance with the legislation and by-laws in this field, has been implemented in Serbia since 2012.For ecological status/potential assessment of a large number of water bodies from biological quality elements were used algae (phytoplankton and benthic diatoms) (KRIZMANIĆ et al., 2013(KRIZMANIĆ et al., , 2015;;VIDAKOVIĆ, 2013;DENIĆ et al., 2014aDENIĆ et al., , 2014bDENIĆ et al., , 2015bDENIĆ et al., , 2015c The aim of this study was to assess the ecological status of the Despotovica River based on biological (epilithic diatoms and aquatic macroinvertebrates) and supporting physico-chemical water quality elements, recommended by National regulation (ANONYMOUS, 2011b).

Study area
The Despotovica River is located in central Serbia (Figure 1a).It springs in southern slopes of the Rudnik Mountain, under the name Rudnik River.It connects with the Majdan River in Majdan village and builds the Despotovica River.The Despotovica River runs between Vujan and Ilijak, creating a gorge between Gornji Milanovac and Brđani.In the village of Brđani, after leaving the gorge, the Despotovica River flows into the Dičina River, which through the Čemernica River flows away to the Zapadna Morava River.The area of the Despotovica basin is 220 km 2 , the length of the stream is 24 km, and the average flow rate at the mouth is 0.5 m 3 /s.It belongs to the Black Sea Basin (DRINJAKOVIĆ et al., 2015).
Considering the waste water entry from the flotation of the lead, copper and zinc mine in the Rudnik mountain, as well as the technological and communal waste water of Gornji Milanovac town, the Despotovica River belongs to the group of polluted rivers of Serbia (GAVRILOVIĆ AND DUKIĆ, 2002;DRINJAKOVIĆ et al., 2015;SIMIĆ et al., 2015).

Collecting samples and accompanying location data
Investigation of the Despotovica River was performed in April 2017 at five sampling sites (L1 -above the tailings pond; L2 -under the tailings pond; L3 -above the "Foka" factory; L4 -Mlakovac; and L5 -Brđani under the landfill) (Figure 1b).Biological (phytobenthos and macroinvertebrates) and supporting physico-chemical water quality elements were followed.

Phytobenthos
At all five localities of the Despotovica River, from phytobenthos community, macroalgae thalli and epilithic diatoms were collected.Sampling of epilithic diatoms was performed according to following standard (EN 19346, 2015).All collected samples were immediately preserved in 4% formaldechyde.Macroalgae were observed on temporary preparations.For treatment of the epilithic diatom samples standard method with concentrated sulfiric acid was used (KRAMMER and LANGE-BERTALOT, 1986).Microscopic examination of the permanent slides was done using the light microscope Motic B310 with digital camera BRESSER (9MP) and MicroCamLab software.Identification of the present taxa was done using the appropriate literature (KRAMMER and LANGE- BERTALOT, 1986BERTALOT, , 1988BERTALOT, , 1991;;WEHR and SHEATH, 2003;KOMÁREK and ANAGNOSTIDIS, 2005).Besides to above literature, on-line identification keys were used as additional resources (SPAULDING et al., 2010;KOCIOLEK, 2012).Qualitative and quantitative analysis of epilithic diatoms was performed according to following standard (EN 14407, 2015).Quantitative analysis was based on the relative abundance of identified taxa, represented by valve percentage of each taxon relative to 400 counted valves on each permanent slide.Based on qualitative and quantitative analysis of epilithic diatoms we calculated diatom indices using OMNIDIA software (LECOINTE et al., 1993).

Aquatic macroinvertebrates
Aquatic macroinvertebrate samples were collected according to AQEM protocol.Benthic samples were taken with a 0.0625 m 2 Surber sampler of 250-µm mesh.Samples were preserved using 70% ethanol solution and analysed in laboratory.The study included qualitative and quantitative analysis of the benthic community.Qualitative analysis was done using a binocular microscope (NIKON SMZ800) and identification keys (BELFIORE, 1983;HRABINOVÁ and BITUŠÍK, 2003;MAGAN, 1955;MANN and WATSON, 1964;ROYKOŠNÝ, 1980).Quantitative analysis was carried out by the absolute counting of individuals of each present species.

Ecological status assessment
Ecological status assessment of the Despotovica River is based on a threshold value of status classes for biological (epilithic diatoms and macroinvertebrates) and physico-chemical water quality elements, according to Serbian national regulation on parameters of ecological and chemical status of surface waters and parameters of chemical and quantitative status of ground waters (ANONYMOUS, 2011b).An assessment of ecological status was given based on the worst evaluated parameter of biological and supporting physico-chemical water quality elements.
For saprobiological analysis, a list of bioindicator organisms according to Moog was applied (MOOG, 1995).Due to a complex identification process, as well as low level of confidence, certain taxa were not identified to the species level.The metrics calculation was performed using ASTERICS software (AQEM, 2002), and the indicative ecological status assessment was performed in accordance with the national legislation (ANONYMOUS, 2011b), based on the class boundaries for rivers Type 3 (small and medium watercourses, altitude up to 500 m), as presented in Table 5.
The largest number of identified taxa belongs to the Ephemeroptera group, while the smallest number was recorded in Crustacea-Amphipoda, Hirudinea and Oligochaeta groups.Of the total 25 taxa, the largest number was recorded at L1 (12 taxa), while the smallest number (2 taxa) was recorded at L2.The most dominant taxon, in relation to the number, was Limnodrilus sp., while Chironomus sp. was present in 80% of the samples.

Analysis of physico-chemical properties of water
Within the research of the Despotovica River, basic physical and chemical parameters of water were followed and analysed, since they have an important influence on the composition and structure of communities in all aquatic ecosystems.The values of measured physical and chemical parameters are shown in Table 3.

Ecological status assessment
According to the Serbian National Regulation (ANONYMOUS, 2011b) the Despotovica River belongs to the water bodies of the Type 3 -small and medium watercourses, altitude up to 500 m, dominance of a large substrate.
Based on analysis of epilithic diatom community parameters, prescribed by the Serbian National Regulation (ANONYMOUS, 2011b), indicative ecological status of the Despotovica River could be assessed as poor (IV class) (Table 4).Down the Despotovica River, indicative ecological status assessment, based on parameters of epilithic diatom community, ranged from excellent (I class -L2) to poor (IV class -L3, L4 and L5).Values of the CEE index were not calculated at L1 and L2, while the value of the IPS index at L1 corresponded to the moderate ecological status (III class) (Table 4).
The results of many authors confirmed that the ecological status assessment based on the IPS index is more trustworthy (VIDAKOVIĆ, 2013;VASILJEVIĆ et al., 2014VASILJEVIĆ et al., , 2016VASILJEVIĆ et al., , 2017;;JAKOVLJEVIĆ et al., 2016b;PREDOJEVIĆ, 2017;VASILJEVIĆ, 2017), primarily because the IPS index usually integrates most of diatom species identified in the sample, as opposed to the CEE index.It was also noted that there was no difference in the boundary values between classes for the CEE index and Vasiljević suggests moving the boundary values for classes for types of watercourses (VASILJEVIĆ, 2017).According to Serbian National Regulation (ANONYMOUS, 2011b) only IPS index is used for assessment of the ecological potential for artificial and significantly modified water bodies (ČAĐO et al., 2016(ČAĐO et al., , 2017;;DENIĆ et al., 2014aDENIĆ et al., , 2014bDENIĆ et al., , 2015bDENIĆ et al., , 2015c)).
Our research confirmed that the credibility of diatom indices depends on the number of taxa used for calculation (LECOINTE et al., 1993).As a consequence of using a small number of taxa, there were differences in estimation of the ecological status classes at the localities down the Despotovica River, based on aforementioned indices.In our research, values of the IPS index pointed to worse class than the values of the CEE index.At L1 and L2 sampling sites, the CEE index was not calculated due to small number of recorded taxa (Table 4).
At the L2, of a total of 4 recorded taxa (Achnanthidium microcephalum, Amphora veneta, Gomphonema olivaceum and Nitzschia palea), A. microcephalum (85.57%) was the most frequent (Table 1).At this locality, the presence of teratological forms of A. microcephalum and N. palea was recorded.A. microcephalum is a cosmopolite that has a wide ecological valence and occurs in all types of water, from oligotrophic to eutrophic (KRAMMER AND LANGE-BERTALOT, 1991).The presence of teratological forms of diatoms may indicate an increase in heavy metals in water (VASILJEVIĆ, 2017; OLENICI et al., 2017).Sampling site L2 is located near the tailings pond of floatation of lead, copper and zinc ore.Teratological forms of A. microcephalum and associates are also found in waters loaded with lead and copper (OLENICI et al., 2017).Potential presence of heavy metals in the Despotovica River submitted (ANONYMOUS, 2011b) diatom indices are not able to detect, since they are designed to assess primarily organic pollution and nutrient loads.
High level of organic pollution on each locality, except L2, was also confirmed by recorded macroalgae.Based on saprobic list of indicators according to Sládeček (SLÁDEČEK, 1973), recorded species on these localities are indicators of exactly the same saprobic degree which could correspond to the classes of ecological status assessment.Cladophora glomerata, recorded at L1, on which the indicative ecological status is estimated as moderate (III class), is an indicator of β-mesosaprobic water quality.Stigeoclonium tenue and Ulothrix zonata are indicators of α-mesosaprobic water quality, which corresponds to poor (IV class) ecological status assessment obtained by diatom indices at L3, L4 and L5.Macroalgae as a biological quality element are not included by the Serbian National Regulation (ANONYMOUS, 2011b).
Based on analysis of aquatic macroinvertebrate communities, indicative ecological status of the Despotovica River could be assessed as bad (V class) (Table 5).
Several authors have discussed relations between the community structure and water status (WASHINGTON, 1984;ROSENBERG and RESH, 1993;CHAPMAN, 1996;SIMIĆ, 1996).In order to achieve an effective ecological status assessment, with high confidence, several metrics should be used.
Indicative ecological status, based on the parameters of aquatic macroinvertebrate community was assessed as bad at all localities, except at L1, where it was assessed as poor (Table 5).
A small number of identified species with the dominance of one or several species indicates stress in aquatic ecosystem.The mass development of Oligochaeta (Limnodrilus sp.) at L4, followed by the numerical reduction of the other macroinvertebrate representatives, indicates the presence of excessive organic matter and bad ecological status (Table 5).Next to these parameters, and in accordance with the national legislation (Official Gazette of the Republic of Serbia 74/2011), the Balkan Biotic Index (BNBI) was also used (SIMIĆ and SIMIĆ, 1999), representing a combination of diversity index and characteristic dominant families, genera and characteristic species of macroinvertebrates only.
The values of BNBI index recorded in the localities L1 and L3 correspond with the Class IV (poor status).The remaining localities also had records of low values of the Class V index (bad status), which indicates a heavy load of organic matter in the water.
The SI values recorded at the Despotovica River show that p-mesosaprobic and αmesosaprobic bioindicators prevail, indicating a high presence of organic pollution and pointing to bad ecological status (Class V).
After taking into consideration all the parameters mentioned above, the indicative status of the Despotovica River could be assessed as bad (Class V), which corresponds to the water quality assessment based on the evaluation provided by national water quality monitoring for the period 2009-2010 (DENIĆ et al., 2015b), which generally assessed the Despotovica River as belonging to Class IV-V.
The assessment of the ecological status based on physico-chemical parameters of water is shown in Table 6.
Increased concentration of nutrients at localities influenced the ecological status of the Despotovica River based on physico-chemical parameters to be assessed as bad (V class) (Table 6).As physical and chemical parameters were measured one time, they can only preliminary indicate the abiotic conditions in this river.Overall ecological status estimation of the Despotovica River in April 2017, based on parameters of biological (epilithic diatoms and aquatic macroinvertebrates) and supporting physico-chemical water quality elements, could be assessed as bad (V class) (Figure 2).Since all relevant parameters of recommended quality elements have been used in assessment, except specific pollutants, and that the frequency of monitoring is lower than the minimum predicted, the level of reliability of the ecological status assessment is medium (WFD, 2000).

CONCLUSION
Indicative ecological status of the Despotovica River in April 2017, based on condition of epilithic diatoms and macroinvertebrate communities, as well as physicochemical parameters of water, was assessed as bad (V class).The bad ecological status at L3, L4 and L5, based on the parameters of epilithic diatoms and aquatic macroinvertebrate communities, confirms the fact that these localities are under the influence of strong organic pollution.
Our research confirms the fact that diatom indices IPS and CEE are good indicators of organic and inorganic pollution.At L2, ecological status based on IPS index was assessed as high, but the presence of teratological forms of diatoms indicates an increased presence of heavy metals at this locality.
The results of our research once again confirm that the IPS diatom index provides a more reliable assessment than CEE diatom index.
For a more precise picture of the ecological status of the Despotovica River, it has been recommended to carry out the monitoring at least four times a year and at more localities down the river.Also, all predicted parameters of biological (phytobenthos, macroinvertebrates, macrophytes and fish) and physico-chemical quality elements should be used for the final assessment of the ecological status.

Figure 1 .
Figure 1.a) Position of the Despotovica River in Serbia; b) Sampling sites of the Despotovica River.
L2 and L3) to 2.24 (α-β-mesosaprobity; L1) and 3.41 (polysaprobity; L4) which indicate presence of organic pollution.These values correspond to Class V (bad status).The lowest diversity was observed in the Despotovica river (Shannon-Weaver Diversity Index values ranging from 2.03 to 0.63 indicate an overall poor water quality (Class IV).Number of Ephemeroptera, Plecoptera and Trichoptera (EPT) taxa ranged from 9 (locality L1) to 0 (L5).Average values of EPT taxa correspond to Class V (bad status).Calculated values of BMWP Score were in the range from 90 (L1) to as little as 8 (L5).Such BMWP Score values indicate bad status of the river (Class V).The highest ASPT value was 10 (L2) and the lowest was 2.6 (L5) (bad status).

Figure 2 .
Figure 2. Overall ecological status assessment of the Despotovica River in April 2017.

Table 1 .
Qualitative and quantitative analysis (percentages number of valves) of epilithic diatoms from the Despotovica River in April 2017.

Table 2 .
Qualitative and quantitative analysis (absolute number of individuals) of macroinvertebrates from the Despotovica River in April 2017.

Table 3 .
The physical and chemical parameters of water in research localities of the Despotovica River in April 2017.

Table 4 .
Ecological status assessment (ESA) of the Despotovica River based on epilithic diatom community in April 2017.

Table 5 .
Ecological status assessment (ESA) of the Despotovica River based on aquatic macroinvertebrate community in April 2017.Values of the Zelinka and Marvan Saprobic Index ranged from 2.1 (β-mesosaprobity;

Table 6 .
Ecological status assessment (ESA) of the Despotovica River based on physico-chemical parameters in April 2017.