The study area is located in the upper part of the Iskar River before the Iskar Dam in Northern Rila, Bulgaria. Iskar is the longest (wholly) Bulgarian river (368 km) with a river basin of 8 650 km². The study river sector is 10 km in length, and 25–35 m in width; its depth ranges from 50 to 200 cm and the bottom substratum consists of pebbles, coarse and medium sands. The seasonal character of flow is determined with summer and winter low flow (1–3 m³/s), a little increase in flow during the autumn (6–10 m³/s) and very expressive spring high water level (15–25 m³/s). According to a map of the land use, agricultural land, pastures and forests dominate the area (Topalova et al. 2013; Todorova et al. 2017). In this river sector, one significant source of point pollution is registered – the discharge from WWTP Samokov (44 100 m³/day). The treatment plant treats the mixed wastewater from the town of Samokov (municipal, industrial and storm) and works on the denitrification/nitrification scheme.

The sampling design included sites upstream and downstream of the WWTP. We carried out two sampling campaigns in November 2020 and March 2021 when the average water flow is 5–8 m³/s. Paired water and sediment samples were collected from four sampling sites (Fig. 1).

Sampling site UI1 – Iskar above WWTP Samokov

Sampling site UI2 – Discharge of WWTP Samokov

Sampling site UI3 – Dragushinovo village, under WWTP Samokov

Sampling site UI4 – Iskar River, near the Villa zone Mechkata (site from monitoring system for surface water bodies in Bulgaria)

We analyzed the physicochemical parameters (temperature, oxygen concentration, conductivity and pH) of the water in situ immediately after sampling with a portable oxygen and pH meters. The hand net was used for collecting the macroinvertebrates – up to 10 sub-samples were collected on one site according to multihabitat approach (EN ISO 10870:2012, EN ISO 16150: 2012). The assessment of the ecological status was conducted by metrics “biotic index” in ranges for river types R4 (semi-mountain rivers in 12 Ecoregion “Pontic Province”). The sediment samples for chemical, microbiological and fluorescent analyses were collected by manual dragging. The all water and sediment samples were transferred in sterile containers at 4 °C storage and processed within 24 h.

The determination of the organic loading in the water was performed by measuring the chemical oxygen demand (COD) by colorimetric dichromatic method. Nitrogen was measured as dissolved inorganic forms – ammonium and nitrate ions (colorimetric methods). The concentrations of phosphorus were defined as phosphates also by use of colorimetric method. Procedural details for measuring nutrients and organics were in line with standard methods recommended in EN ISO standards. The concentrations of selected microelements in the water samples were determined with inductively-coupled plasma mass spectrometry (ICP-MS, PerkinElmer SCIEX Elan DRC-e). The number of the decimal places is related to the precision of the measurements. The estimation of accuracy was conducted by the analysis of two water standard reference materials: SPS-SW2 (Reference Material for Measurement of Elements in Surface Waters, Spectrapure Standards, Norway) and NWTM-23.5 (Environmental matrix reference material, a trace element-fortified sample, Environment and Climate Change, Canada). The experimental results were in very good agreement with the certified values.

Data from chemical analyses of water and assessment of ecological status was compared with the requirements of Bulgarian legislation (Regulation No. H-4 of 14.09.2012 on characterization of surface water and Regulation No. 12 of 18.06.2002 on the quality of surface water intended for drinking and household purpose). Selecting microelements and ranking hazardous and specific pollutants was conducted on the basis of the EU-list of priority substances (Water Framework Directive 60/2000/EC (WFD) and Regulation on environmental quality standards for priority substances and some other pollutants, 2015) and the above-mentioned regulations.

The total microbial count and number of coliforms was determined by the use of standard count-plate technique on Nutrient agar and Lactose TTC Agar with Tergitol 7 (Merck Millipore). The sediment samples were preliminarily treated with ultrasonic disintegrator VCX 750, Sonics & Materials Inc. (3 times × 10 sec). The data for microbial counts were normalized and presented as ln CFU/mL or ln CFU/g dry weight.

For analysis of changes in microbial activity of sediment communities, we used a modification of CTC/DAPI staining method with fluorescence imaging. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) enters in cells and is reduced to CTC-formazan (fluorescent red signal). The content of reduced compound depends on the electron transport activity (activity of the dehydrogenase enzymes in viable cells) and is considered as an indicator of their metabolic activity. DAPI (4′,6-diamidino-2-phenylindole) is fluorescent dye that specifically binds to nucleic acids but enters both in live and fixed cells. It is widely used for the enumeration of bacterial abundance. The combined staining with CTC and DAPI is applied to distinguish the active fraction in different microbial communities (Topalova et al. in press). The epifluorescent microscope Leica DM6 B was used to make fluorescence images, and then a digital image analysis (using the software daime) was applied to assess the total area, the area of fluorescent objects, and mean fluorescence intensity. From these parameters the percent of live, metabolically active cells in images was calculated (Daims et al. 2006; Topalova et al. in press).

In the study area, the water temperature showed typical seasonal dynamics and varied from 4.2 °C at sampling site UI3 to 8.9 °C at sampling site UI2 in November (Table 1). The highest temperature in both seasons was reported in the water from the discharge area of WWTP. The values of pH varied between 7.63 and 8.80 – the highest value was measured at UI2. The content of dissolved oxygen is usually high for this part of the Iskar River because of its rapid flow rate. The lower concentrations in all sampling sites for this parameter were measured in March, when the water level and turbidity were higher as snow melted, and with the onset of the wet season. At the point of WWTP discharge, the oxygen concentration was significantly lower during both samplings and the conductivity was higher than the site located above. In March, the measured values for oxygen and pH in UI2 exceeded the recommended norms for high status but the fluctuations were assimilated downstream.

In Fig. 2 we present the impact of WWTP effluent on the dynamics of nutrients (nitrogen and phosphorus) in surface water for both sampling campaigns (Fig. 2A – November and Fig. 2B – March). With regard to nitrogen, there was a significant impact on concentration of nitrates and ammonium ions from the discharge from the WWTP effluent. The highest values of nitrates were registered in water of sampling site UI2 during the both samplings. The concentration of nitrates increased almost 20-fold in comparison with the previous site in November and 8-fold in March. The situation was similar for ammonium during the autumn – the increase was more than 15-fold. Phosphate concentrations in surface waters were higher in November (between 1.087÷1.358 mg/L) compared to the values observed in March (0.188÷0.750 mg/L). During the autumn, the phosphate concentration retained similar very high levels in the whole river sector. In March, the discharge of WWTP effluent affected the phosphate concentration in the main river channel and the measured value at UI2 site was 0.750 mg/L. The nitrates were in the range acceptable for surface waters, although the WWTP effluent affected their concentration, but the ammonium ions and phosphates exceeded the values admissible according to the stipulations of Bulgarian legislation.

The values ​​for chemical oxygen demand (COD) during the two samplings at three of the four sampling sites were below 10 mgO₂/L. The highest concentration was measured in site UI2 in March – 11.98 mgO₂/L.

The concentrations of the selected microelements in the water samples are presented in Table 2. The data showed an increased level of risk, associated with seasonal dynamics of Hg, being a priority pollutant (Regulation on environmental quality standards for priority substances and some other pollutants, 2015) and Cu – the measured values in November were higher than those measured in March for all sampling sites. The highest mercury concentration was determined in the water of site UI3 (after discharge of WWTP) – 1.42 µg/L; for copper – 44 µg/L was measured at site UI2. The concentrations of Cu increased more than 10–30 times in the autumn. The concentrations of the rest of the microelements were below the admissible values in both seasons. As a result of increased anthropogenic activity in the post-summer period, significant seasonal changes are worth noting in all sampling sites for Zn (till 25 times in UI4), Cr (till 11 times in UI1) and As (till 7 times in UI1). To a certain extent, the same tendency is also established for Fe with the exception of sampling place UI4. The contents of the toxic elements Cd, Ni, Pb, being also priority pollutants, and Mn showed relatively close values in the studied sampling sites and significant stability between the seasons.

The data from determination of total microbial count and coliforms in water and sediments are presented in the figures below (Fig. 3, 4).

In water samples from the studied part of the river, we enumerated stable values for total culturable microflora – in the range of 10³÷10⁴ CFU/mL. The coliforms were constantly presented in water samples with abundance of 10÷10² CFU/mL. According to site location, the numbers of two indicator groups showed a slight increase in the sampling site of WWTP discharge during the each season studied. In the sediment samples, the total microflora was more abundant and variable. The numbers fluctuated between 10⁵÷10⁷ CFU/g. In the sediments of discharge area of WWTP, the increase in numbers of two indicator groups was significant, especially for coliforms (3 000÷11 000 CFU/g).

The mean fluorescence intensity and percent live cells were calculated from the total area and the area of fluorescent objects on images from CTC/DAPI analysis (Table 3). The fluorescence intensity as an indicator of viability and activity of cells was assessed on CTC staining images. It can be seen that the parameter was up to 2.3 times higher in U13 when compared to the other two sites. The metabolic activity of the bacteria in that site remains high also in March of the following year (with up to 35%). The share of live cells was calculated as ratio of bacteria with metabolic activity (CTC) to total bacteria (DAPI). At sampling site UI3, a high share of live cells (2.20%) was also detected in November, but their share decreased by 10.5 times in March. The data for UI2 revealed clear differences in the share of live cells in sediment samples compared to UI1 – the live cells were about 3% in both seasons. The fluorescent images of sediment communities in sampling site UI2 were presented in Fig. 5.

The ecological status of the studied river sector was assessed as “good” according to WFD and Regulation N-4/2012 using macroinvertebrate bioindicators. The metrics Biotic index had a score of 4 in both sites for macrozoobenthic analyses (UI1, UI3). Despite similar ecological status, in sampling site UI1 species tolerant to deterioration in environmental conditions were found – Hydropsyche sp. (Trichoptera), Chironomidae (Diptera), Erpobdella octoculata/monostriata (Hirudinea). After discharge of WWTP Samokov, in sampling site UI3, the total taxa number was higher and Baetis sp. (Ephemeroptera) was also found.