Estuaries are crucial areas for the assessment of eutrophication (HELCOM 1998; Golubkov et al. 2017), because they supply marine waters with nutrients and organic matter, which, in turn, fuel phytoplankton and bacterial productions (Jost & Pollehne 1998; Urrutxurtu et al. 2003; Mironova et al. 2013; Wielgat-Rychert et al. 2013; Rychert et al. 2014; Golubkov et al. 2017). Both phytoplankton and bacteria are food sources for protozoa (heterotrophic protists) that transfer a considerable part of phytoplankton primary production and bacterial secondary production to higher trophic levels, i.e. zooplankton and larval fish (Sherr & Sherr 2002; Rychert et al. 2016). This microbial food web is equally important in freshwater and marine environments (Sherr & Sherr 2002; Weisse 2017).
Gradients of water salinity and concentrations of nutrients and organic matter obviously drive the distribution of organisms. The abundance of protists (algae and protozoa) is high in the inner estuaries, but it drops significantly toward the open sea (Urrutxurtu et al. 2003; Christaki et al. 2009; Mironova et al. 2013; Wielgat-Rychert et al. 2013; Rychert et al. 2014). At the same time, a decrease in the abundance of bacteria is observed, but it is usually less conspicuous (Christaki et al. 2009; Wielgat-Rychert et al. 2013). To date, studies on the abundance of microorganisms along salinity gradients have generally been limited to large estuaries, while gradients along small estuaries remain understudied.
In this work, we studied the estuary of a medium-sized coastal river – the Słupia River, which discharges its waters into the Baltic Sea. Its catchment area covers 1623 km2, its length is 138.6 km, and mean annual discharge is 18 m3 s−1 (HELCOM 1998). Thus, it is much smaller than the largest inflow to the southern Baltic, i.e. the Vistula River, the average discharge of which is 1081 m3 s−1 (HELCOM 1998). The mouth of the Słupia River enters the port in the town of Ustka, which is mainly used by fishing cutters. The length of the port is about 1.1 km. The objective of this study was to assess the distribution of environmental parameters, chlorophyll
The study was conducted on 18 July 2014. During the study, the weather was good with cloud cover of 2–3/8, and the sea state was 3–4°B. No precipitation was observed. Samples were collected from the Słupia River, the port of Ustka, and the marine waters outside the port (Fig. 1). Sampling was performed between 11:00 and 17:00. Samples 1–8 were collected from the surface zone. At sampling site 3, an additional sample (3D) was collected from the near-bottom zone (5.4 m). This sample was collected with a bathometer. Sampling at sites 1–3 was performed from bridges, while at sites 4–8 (outside the port) – from aboard a small cutter. The water depth at sampling site 1 (at the mouth of the Słupia River) was 0.2 m and it ranged from 4.0 to 5.4 m at sites 2–3 (the port). Outside the port, the depth gradually increased from 5.6 m (sampling site 4) to 26.5 m (sampling site 8). At each sampling site, 200 ml of water was fixed with formalin and another 250 ml of water was fixed with acid Lugol’s solution. Additionally, unfixed water was collected for measurements of chlorophyll and total suspended matter concentrations. These measurements were performed a few hours after sampling. Chlorophyll
Bacterial numbers were studied in samples fixed with formalin. Analysis was performed under an epifluorescence microscope after concentrating bacteria onto nucleopore filters and staining them with acridine orange (Hobbie et al. 1977). Ciliates were analyzed in samples fixed with acid Lugol’s solution under an inverted microscope, i.e. using the Utermöhl method (HELCOM 2001). Taxonomic identification was performed according to Marshall (1969), Maeda & Carey (1985), Foissner & Berger (1996), a web-based guide (Strüder-Kypke & Montagnes 2002), and other keys. Generic and species names were verified in accordance with the World Register of Marine Species (WoRMS 2017). Each ciliate cell was measured using a camera and image analysis software to estimate its volume. Cell volume (CV, μm3) was recalculated into biomass (CC, carbon content, pg C) according to the
following formula published by Menden-Deuer & Lessard (2000):
The biomass of loricate ciliates was calculated from the volume of loricae (LV, μm3) with the following formula by Verity & Langdon (1984):
Freshwater residence time for the port of Ustka was calculated as the quotient of water volume and mean annual discharge of the Słupia River. The area of the port is 11.5 ha, but that of the inner port (the area between stations 1 and 3; Fig. 1) is only 5.39 ha (Maritime Office in Słupsk 2012). The depth of the canal and basins in the port ranges from 4.5 to 6.5 m (Konkol & Wickland 2016). The mean annual discharge of the Słupia River is 18 m3 s−1 (HELCOM 1998). Based on the mean depth of the port and the mean discharge of the river, the freshwater residence time computed for the inner port was 4.6 h. This residence time for the whole area of the port (including the outer harbor) was estimated at 9.8 h. Both values are annual means.
Statistical analyses were performed with PAST 3.16 statistical software (Hammer et al. 2001).
The temperature of the surface waters ranged from 19.4 to 20.4°C at all sampling sites. In the near-bottom zone at station 3 (sample 3D, depth 5.4 m), the temperature was lower at 18.1°C. At the mouth of the Słupia River (station 1; Fig. 1), water salinity was 0.21 PSU, and in the port of Ustka (stations 2–3) it ranged from 0.77 to 0.87 PSU. The salinity at the near-bottom zone at site 3 (sample 3D) was much higher at 6.81 PSU, which corresponded to the salinity of the surface waters outside the port (6.61–7.80 PSU, stations 4–8). Two different water masses were identified based on the salinity: (i) less saline water in the river and in the surface layer in the port (samples 1–3), and (ii) more saline water in the near-bottom zone in the port (sample 3D) and in the surface layer outside the port (samples 4–8; Table 1). Within the port, more saline and dense water was below the less saline surface water. The two water masses were also indicated by different water transparencies measured as Secchi depth: 2.5–3.0 m in the port (sites 2–3) and 5.0–7.5 m outside the port (stations 4–8).
Parameters measured in two water masses separated along the transect between the Słupia River mouth and offshore waters. Less saline water was observed in the river mouth and in the surface layer in the port (samples 1–3), whereas more saline water was observed in the near-bottom zone in the port (sample 3D) and outside the port (samples 4–8). Obviously, Secchi depth was not determined at near-bottom sampling site 3D. Statistically significant differences between the two separate water masses were demonstrated for each parameter.
Parameter | Samples 1–3 | Samples 3D, 4–8 | Statistical significance according to Mann-Whitney |
---|---|---|---|
Salinity (PSU) | 0.21–0.87 | 6.61–7.80 | |
Secchi depth (m) | 2.5–3.0 | 5.0–7.5 | |
Bacterial abundance (106 ml−1) | 5.51–6.16 | 0.99–2.14 | |
Ciliate abundance (cells ml−1) | 0.34–0.90 | 2.65–5.40 | |
Ciliate biomass (ng C ml−1) | 0.27–0.75 | 1.46–4.40 |
Bacterial abundance in less saline waters (samples 1–3) ranged from 5.51 to 6.16 × 106 ml−1, whereas in more saline water (samples 3D, 4–8) it was lower and ranged from 0.99 to 2.14
The separation of the water into two masses was demonstrated to be statistically significant with respect to most of the parameters studied (Table 1). However, the pattern of distribution of chlorophyll
It was observed (Fig. 3) that the ciliate community composition reconstructed itself along the transect from the river mouth to offshore waters. In the river (sample 1), the ciliate community consisted of hypotrichs (40% of ciliate biomass), prostomatids (26%), and scuticociliates (14%). At this sampling site, many ciliates classified into the group “other and unidentified” (Fig. 3) were benthic migrants, e.g. hymenostomatids. In the surface waters in the port (samples 2–3), the most important ciliates were prostomatids, which contributed 50–90% of the ciliate biomass. Both in the river mouth and in the surface waters in the port (samples 1–3), oligotrichs and choreotrichs contributed only 3–4% of ciliate biomass (Fig. 3). In the near-bottom zone in the port and in the surface waters outside the port (samples 3D and 4–8, more saline waters), the most important were oligotrichs and choreotrichs, the contribution of which to ciliate biomass ranged from 45 to 80%. The remaining biomass (Fig. 3) consisted mainly of prostomatids (5–37%) and haptorids (up to 14%). The difference between the contribution of oligotrichs and choreotrichs to ciliate biomass observed in (i) less saline waters (3–4% of ciliate biomass) and in (ii) more saline waters (45–80% of ciliate biomass) was statistically significant (Mann-Whitney
Among the prostomatids observed in less saline waters (samples 1–3), the most frequent were those from the genera
Detailed data on abundance, biomass, and composition of ciliate communities were provided in the supplementary material.
Outside the port of Ustka, water salinity and Secchi depths corresponded to typical values observed in the southern Baltic Sea (Matthäus et al. 2008; Fleming-Lehtinen & Laamanen 2012). Water transparency in the port was lower because chlorophyll concentrations were higher (Fig. 2). The concentrations of chlorophyll
The bacterial abundance reported in this study was lower than that in the eutrophic Warnow River (southwestern Baltic Sea, July, 10–20 × 106 ml−1; Freese et al. 2006), but it corresponded well to abundance observed during summer in estuaries and coastal waters of the Baltic Sea (Mironova et al. 2012; Wielgat-Rychert et al. 2013; Rychert et al. 2013; Rychert et al. 2015) and other waters (Christaki et al. 2009). For example, in the early summer the bacterial abundance in the Vistula River was 6.87 × 106 ml−1 and in the offshore waters of the Gulf of Gdansk (Baltic Sea) it was on average 1.82 × 106 ml−1 (Wielgat-Rychert et al. 2013).
The ciliate abundance in the river mouth and in the surface waters in the port was low compared to the abundance observed previously in July in the Słupia River (mean 4.00 cells ml−1, 4.35 ng C ml−1; Rychert 2009). The ciliate abundance observed in this study in more saline waters was also lower than that reported in July 2006 and 2007 in coastal waters located 1.3 km to the west of the port of Ustka at 6.01–46.5 cells ml−1 and 3.69–71.87 ng C ml−1 (Rychert et al. 2016). The ciliate abundance observed in this study was comparable to values observed in the Neva River estuary (Baltic Sea) in July 2008 (about 3 cells ml−1 and 5 ng C ml−1; Mironova et al. 2012), and it was higher than that observed there during summer 2010 (0.03–1.9 cells ml−1; Mironova et al. 2013).
In large estuaries, e.g. the Vistula River estuary (Baltic Sea; Wielgat-Rychert et al. 2013) and lagoon and coastal waters of the Pomeranian Bight (Baltic Sea; Jost & Pollehne 1998), gradual decreases in chlorophyll
In contrast to phytoplankton (chlorophyll
Ciliates observed in less saline waters (the river mouth, surface waters in the port) were previously reported in the Słupia River (Rychert 2009) and other Baltic rivers (e.g. Griniene et al. 2011), whereas ciliates encountered in more saline waters (near-bottom waters in the port, surface waters outside the port) were reported in the Baltic Sea (Griniene et al. 2011; Mironova et al. 2012; Mironova et al. 2013; Rychert et al. 2013; Mironova et al. 2014). The reconstruction of the ciliate community observed in this study generally resembled the changes in the ciliate community composition previously described along large Baltic estuaries, e.g. the Neva estuary (the Gulf of Finland; Mironova et al. 2012), and the Vistula River estuary (the Gulf of Gdansk; Rychert et al. 2014). Ciliate communities in the inner part of these estuaries contain some freshwater species and benthic migrants. In the outer parts of the Neva and the Vistula estuaries, ciliate communities are dominated by oligotrichs and choreotrichs (including tintinnids) and are accompanied by haptorids (Mironova et al. 2012; Rychert et al. 2014). The same dominants in the ciliate community, but with prostomatids, were observed in this study in more saline waters (samples 3D, 4–8). We observed that the importance of prostomatids was considerable since they contributed 26–90% of the ciliate biomass in less saline waters (stations 1–3) and 5–37% of the ciliate biomass in more saline waters (samples 3D, 4–8). This corresponds well to the previously demonstrated high importance of prostomatids in the Słupia River, where they contribute 41% of the ciliate biomass annually (Rychert 2009), and the lesser importance of prostomatids in the coastal waters near Ustka, where they contribute only 13% of the ciliate biomass annually (Rychert et al. 2013, unpubl. details). In some estuaries, e.g. that of the Nervión River (the Bay of Biscay, Atlantic Ocean; Urrutxurtu et al. 2003), ciliate communities were dominated by scuticociliates, which was explained by the high availability of bacteria that are their food resources (Urrutxurtu et al. 2003). In this study, bacterial abundance was lower and scuticociliates were observed only in small quantities. Mironova et al. (2012) reported that in the Neva estuary (Baltic Sea) in summer, 28–67% of ciliates were mixotrophs. A significant presence of mixotrophs was also reported in estuaries located in other seas, e.g. in the Rhone River estuary (Mediterranean Sea; Christaki et al. 2009). In this study, ciliates were analyzed in samples fixed with Lugol’s solution, which made it impossible to observe the chloroplasts inside the cells. Thus, we did not estimate the fraction of mixotrophs in the ciliate community. Among easily identifiable marine mixotrophs (
Two different water masses were identified based on the majority of parameters studied (water salinity, Secchi depth, bacterial abundance, ciliate abundance, ciliate biomass): (i) less saline water in the river and in the surface layer in the port, and (ii) more saline water in the near-bottom zone in the port and in surface layer outside the port. The distinct separation of the two water masses, i.e. the absence of gradual gradients of these parameters, indicated that the Słupia River exerted a minor impact on the marine waters. Thus, the hypothesis presented in the introduction was generally confirmed. Only in the case of chlorophyll