A complex biofilm that develops on submerged substrates represents a heterogeneous community referred to as periphyton. Periphyton consists of both autotrophic and heterotrophic (organic) components as well as an inorganic component originating from different kinds of particles from the water column. The autotrophic component of periphyton is mainly composed of algae and Cyanobacteria, while the heterotrophic component is comprised of bacteria, fungi and microinvertebrates. This complex assemblage plays an essential role in the basic processes of the ecosystem – primary production and nutrient cycling, as well as particle and pollutant filtration (Azim et al. 2005). The development of periphyton is driven by the complex interactions between various abiotic (hydrological regime, light, nutrients) and biotic factors (Albay, Akcaalan 2008). Since algae are the most diverse and abundant fraction of periphyton, this complex community is a potential source of valuable information on the environmental state of surface waters (Borduqui, Ferragut 2012). A complex interaction between environmental parameters, the primary nutrient level, light conditions, colonization time and the substrate type affects the development of the periphyton community (Zhang et al. 2013). The potential of organic substrates as an additional source of nutrients and the way they affect the periphytic community are emphasized (Zhang et al. 2013).
Various artificial substrates (both of inorganic and organic origin) are employed in both the periphyton ecology and biomonitoring studies (B-Béres et al. 2016; Stenger-Kovács et al. 2013; Zhang et al. 2013; Žuna Pfeiffer et al. 2015), neglecting the effect of the substrate type on the development of periphyton (Cattaneo, Amireault 1992; Schevchenko 2011). Usually, the significance of dissimilarities between communities developed on natural and artificial substrates is pointed out, but very few studies paid attention to potential differences between communities developed on different kinds of artificial substrates, especially when wooden or similar (organic origin) and inorganic types of substrates are used (Sabater et al. 1998; Danilov, Ekelund 2001; Zhang et al. 2013).
The ecological importance of algal growth forms (ecological groups) has been studied since Passy (2007) introduced diatom ecological guilds. However, DeNicola et al. (2006) also emphasized the significance of algal growth forms, but they took into consideration both diatoms and soft bodied representatives of the periphytic algal community. The bioindication capacity of ecological groups has been confirmed in many studies (DeNicola et al. 2006; Passy 2007; Stenger-Kovács et al. 2013; B-Béres et al. 2016). Stancheva & Sheath (2016) postulated that although diatoms are preferred in biomonitoring studies (studied per se, excluding non-diatom algae in the community), soft bodied algae have a great potential and should be reconsidered as bioassessment tools. To summarize the literature, Tapolczai et al. (2016) also emphasized the importance of considering non-diatom algae in functional classification.
Periphyton colonization and successional trajectories are driven by integrated allogenic and autogenic factors, but the complexity of these processes in periphytic biofilm makes the determination of the main factors imprecise (França et al. 2011). Hydrological disturbances, the nutrient level and seasonality were emphasized as the factors that strongly affect the taxonomic and functional composition of the periphytic community and successional patterns (França et al. 2011; Dunck et al. 2015; Larson, Passy 2012). Only one study assessed the effect of the substrate type on periphytic algal life forms, but only in terms of the substrate roughness, and it was concluded that the intensity of disturbance was the main driver of periphytic algae settlement processes and predominantly determined the effect of substrate heterogeneity (Schneck, Melo 2012).
In our study, we deployed four kinds of artificial substrates (two inorganic ones – glass and ceramic, and two organic ones – willow and yew wooden tiles) for the development of periphyton in an urban reservoir referred to as Lake Savsko (Belgrade, Serbia). We comparatively investigated the structure, colonization process, diversity and successional trajectories of periphyton developed on different kinds of substrates, in order to determine whether the type of substrate affects these periphyton characteristics. We also assessed the relationship between the dynamics of algae growth forms (ecological groups) on different substrates and ecological factors in order to determine whether an artificial substrate potentially affects the capacity of ecological groups to indicate changes in environmental conditions.
Lake Savsko (44°47’02.28”N, 20°23’25.64”E; 73 m a.s.l) is the largest urban reservoir in Belgrade and plays an important role in supplying drinking water (through bank filtration) to approximately 2 million people, and is used for recreational purposes. Lake Savsko was formed by embanking the Sava River near its confluence with the Danube. From the upper, southwestern side, the lake is refreshed by the infiltration of clarified water from a sedimentation unit. The pumping station at the downstream side enables a suitable water level and flow control. It is well connected with public transport, pedestrian and cycling lanes, and more than 100 000 people visit the reservoir every day at the peak of the summer season (Mićković et al. 2014). The reservoir is partially surrounded by vascular vegetation composed of white willow (
During the summer period (July to September) of 2014, artificial substrates (glass, ceramic, willow and yew tree tiles) were submerged into the photic zone of Lake Savsko (11 July), at a depth of 50 cm from the water surface, using an acrylic holder as a carrier. The acrylic holder was attached to a floating buoy that was anchored in the northeastern part of Lake Savsko (Fig. 1). All tiles had uniform dimensions, 2.6 × 7.6 cm, and were vertically positioned in the water column. Periphyton samples were collected weekly, from 20 July to 9 September (after 7-9 days of exposure, altogether 8 sampling weeks), and always in triplicate for each type of substrate.
Location of Lake Savsko and the sampling siteFigure 1
Water transparency was measured in situ using a Secchi disk, while water temperature and dissolved oxygen/saturation were measured using a YSI ProODO Optical Dissolved Oxygen Instrument. Water samples for chemical analyses were collected under the water surface using a Ruttner bottle and transported to the laboratory at the Institute of Public Health of Serbia, where all analyses were performed using standard analytical methods (APHA 1995). Chlorophyll
Substrates with developed periphyton were collected from 9 a.m. to 1 p.m., stored in separate sterile plastic containers and transported to the laboratory in a mobile freezer. In the laboratory conditions, the periphyton from the upper surface of each tile (19.76 cm2) was scraped using a stainless steel razor blade, suspended in 100 ml of tap water and homogenized with a hand blender. From each suspension, 3 subsamples were taken. The first subsample of 40 ml for taxonomic and quantitative analyses was preserved in 4% formaldehyde (final concentration) according to the Guidance Standard on the Enumeration of Phytoplankton using Inverted Microscopy (Utermöhl Technique), (EN 15204 2006). The second subsample of 30 ml was taken for Chl
For taxonomic and quantitative analyses, subsamples from all three replicates (for each substrate type) were merged. The quantitative analyses were conducted according to the Utermöhl method (EN 15204 2006) using a Leica DMIL inverted microscope and the data were expressed as a number of individuals (colonies and filaments considered as one individual) per cm2 (ind. cm2). Six ecological groups were defined based on the results obtained for ind. cm2, i.e. ecological guilds and growth forms. Diatoms were classified into ecological guilds (according to their growth morphologies): the low profile, the high profile and the motile guild according to Passy (2007), Passy & Larson (2011), Rimet & Bouchez (2011) and Gottschalk & Kahlert (2012). Soft bodied representatives were classified into growth forms: unicellular and colonial (both Chlorophytes and Cyanobacteria, and also one representative of Chrysophyta and three representatives of Dinophyta, which made a very small portion), filamentous Chlorophytes and filamentous Cyanobacteria, according to DeNicola et al. (2006). All taxonomic analyses were performed using a Carl Zeiss AxioImager M1 microscope and a digital camera AxioCam MRc5 with AxioVision 4.8 software. Part of the material was acid treated and mounted on Naphrax for diatom taxonomic verification (Acker et al. 2002). Taxonomic identification was carried out according to the standard literature. The Shannon diversity index (H) (Shannon, Weaver 1949) and Pielou’s evenness index (Eh) (Pielou 1969) were calculated.
The measurements of Chl
In order to define the composition of periphyton, the index proposed by Lakatos was employed (Lakatos 1989). It is a descriptive index based on chlorophyll-a % (Chl type), ash content in % of dry mass (AC type), and dry mass in g m-2 (DM type). It classifies the periphyton into a few categories, i.e. types, from autotrophic to heterotrophic (Chl type I-IV), from inorganic to organic ones (AC type I-IV) and from high to low biomass (DM type I-III).
Coefficients (r) and significance (p) of the correlation between parameters were analyzed by Pearson’s correlations. All analyses were performed using the statistical package Statistica 6.0 (Statsoft, Inc., Tulsa, OK, USA) with a significance level of
The redundancy analyses were performed using the program CANOCO for Windows, Version 5.0 (ter Braak, Šmilauer 2012). For the project’s data, the number of individuals per cm-2 (ind. cm-2) of all recorded taxa was used as a measure. The identified taxa were first grouped into appropriate ecological guilds: the low profile guild (LPG), the high profile guild (HPG), the motile guild (MPG), and the appropriate growth form group (unicellular and colonial, OCC), filamentous Cyanobacteria (CF) and filamentous Chlorophyta (CHF). Then, the potential effects of the measured environmental factors on these groups were examined using RDA. We used 12 explanatory variables (turbidity, silicon dioxide, permanganate index, biological oxygen demand, pH value, oxygen saturation, nitrate content, nitrite content, ammonium ion, conductivity, total phosphorus and orthophosphates) and two supplementary variables (weeks of sampling and substrates). The explanatory variables were submitted to the interactive forward selection where the statistical significance of each variable was tested by the Monte Carlo permutation test at a cutoff point of
During the study period, water temperature varied in a relatively small range, from 24°C to 27°C. Water transparency values recorded in a narrow range, from 2.60 to 3.50 m, point to a relatively low phytoplankton primary production and low turbidity of Lake Savsko. The water was rich in dissolved oxygen – all measured concentrations were above 9.1 mg l-1, while oxygen saturation varied between 82 and 97%. Lake Savsko had alkaline water with a relatively low content of dissolved minerals; conductivity varied in a range of 214-229 μS cm-1. The permanganate index, as a measure of organic load, varied between 5.40 and 11.70 mg l-1. The concentration of silicon dioxide ranges from 0.09 to 0.70 mg l-1. There were slight differences in the ammonium content, with the maximum value determined in the 7th sampling week (Fig. 2a), and in the concentration of nitrites, the values of which were generally low, with the maximum recorded on the start date of the experiment (presented on secondary axes, Fig. 2a), while the highest values of nitrates were recorded in sampling weeks 1 and 4 and were otherwise constantly low (Fig. 2a). Variations in orthophosphates and total phosphorus were within a wide range, 0.001-0.165 mg l-1 for orthophosphates and 0.009-0.198 mg l-1 for total phosphorus, the maximum values were determined on the 6th sampling week, and high values were recorded also at the very beginning (weeks 2 and 3) and at the end of the experiment (weeks 7 and 8) (Fig. 2b). Large variations in the nutrient concentration could be related to the location of Lake Savsko in the urban territory, where swimmers, restaurants and surface runoff (highway and surrounding facilities) are considered to be the main sources of nutrients and pollutants. Our results could also lead to the assumption that the external input of nutrients (promoted by urbanization of the lake’s shore and a large number of visitors) might exceed the capacity of the processes that have a positive effect on the water quality. The disruption of this fragile equilibrium would certainly lead to a failure in achieving the good ecological potential of Lake Savsko. The maximum biological oxygen demand (BOD) was 6.1 mg O2 l-1
Temporal variation of nutrients in Lake Savsko in summer 2014Figure 2
The results obtained using Carlson’s TSI in summer 2014 are presented in Fig. 3. Values of TSI (SD) and TSI (Chl
Carlson’s trophic state index of Lake Savsko in summer 2014Figure 3
The biomass of the developed periphyton was generally low. According to the Lakatos index (Table 1), the periphyton community belonged to the DM type III, except for the community developed on glass during the last three weeks and on ceramic during the last two weeks, when the community was classified as DM type II, which points to the medium level of biomass. Romanów & Witek (2011) reported that the biomass of periphyton from three different types of lakes in Poland also belonged to DM type III, when the period from April to November was considered. The ash content on all substrates (Table 2) in our study indicated that the periphyton structure was prevalently inorganic/organic (AC type II), occasionally organic/inorganic (AC type III) and purely organic (AC type IV). The inorganic nature of the developed biofilm could reflect a relatively high concentration of suspended particles in water (Kiss et al. 2003), originating from various sources and caused by various factors, including rainfall events (according to the Serbian Hydrometeorological Institute 2015, summer 2014 was extremely rainy) and the urban character of Lake Savsko (the largest recreational and swimming center in Belgrade during the summer season). Romanów & Witek (2011) related the ash content in periphyton to the trophic status of the surveyed lakes. In the above mentioned study, the variability in the ash content between categories II, III and IV was basically characteristic of a shallow eutrophic lake (Lake Gardno), while exclusively organic categorization (AC type I) of periphyton was determined in a soft water mesotrophic lake (Lake Maly Borek). Therefore, it can be concluded, based on our results, that the ash content index (Lakatos index) is a good descriptor of local environmental conditions in Lake Savsko. The classification according to Chl
Lakatos index – classification of the developed periphyton according to the values obtained for DM, AC and Chl
Week
Glass tiles
Ceramic tiles
Willow tiles
Yew tiles
DM type
AC type
Chl type
DM type
AC type
Chl type
DM type
AC type
Chl type
DM type
AC type
Chl type
1
III
II
III
III
IV
IV
III
III
IV
III
II
IV
2
III
IV
IV
III
II
III
III
II
IV
III
II
III
3
III
II
II
III
II
II
III
IV
III
III
II
II
4
III
III
III
III
II
III
III
II
III
III
III
III
5
III
III
IV
III
II
III
III
II
II
III
III
III
6
II
II
III
III
II
III
III
II
IV
III
II
IV
7
II
II
IV
II
II
IV
III
II
III
III
II
IV
8
II
II
III
II
II
III
III
II
III
III
II
III
Table 2 shows maximum, minimum and average values for DM, AFDM and Chl
Values obtained for DM, AFDM and Chl
Glass tiles
Ceramic tiles
Willow tiles
Yew tiles
DM g m-2
AFDM g m-2
Chl
DM g m-2
AFDM g m-2
Chl
DM g m-2
AFDM g m-2
Chl
DM g m-2
AFDM g m-2
Chl
max
27.83
9.50
9.09
22.77
9.95
9.09
16.87
7.87
10.29
18.56
6.52
6.86
min.
3.37
0.84
0.86
1.69
0.84
0.34
3.37
1.52
0.34
2.53
0.90
0.34
average
11.49
4.41
6.11
11.07
4.00
4.76
8.65
3.87
5.29
8.54
3.57
4.22
Based on the results obtained for the photosynthetic component (Chl
Periphyton photosynthetic component (Chl a) growth rate dynamics on glass (a), ceramic (b), willow (c) and yew (d) tilesFigure 4
According to literature data, the colonization time of periphyton is usually estimated between 2 and 4 weeks, after which the maximum biomass is reached and sloughing is avoided (Cattaneo, Amireault 1992). In our study, the highest biomass values occurred in weeks 6, 7 and 8 (Table 2), but according to the general pattern of short-term biomass accruals (Azim, Asaeda 2005) and Fig. 4, these values were recorded during the biomass increment observed in fluctuation phase and followed the previous biomass loses. Thus, the overall peak in biomass recorded in our study could be attributed to the recolonization processes and the increase in the community complexity in the latter successional phase, instead of the initial accrual of biomass. This suggests that the real first peak of biomass actually occurred during the first three weeks of incubation, as Biggs (1996) speculated it to be a pattern in conditions of low to moderate disturbance intensity, where the colonization process is promoted by abundant propagules in the refugium.
Algal diversity (H) and evenness (Eh) of the periphytic community developed on each substrate are presented in Fig. 5 (a, b, c, d). It appears that the trend in diversity during the colonization is very similar on both wooden substrates (r = 0.884,
Shannon’s diversity index (H) and Pielou’s evenness index (Eh) on glass (a), ceramic (b), willow (c) and yew (d) tilesFigure 5
On the other hand, communities developed on glass and ceramic were characterized by specific patterns in the diversity dynamics during the colonization time (Fig. 5a, b). The highest values of the H index on both substrates were determined in weeks 6 and 8, while drastic declines in the diversity were determined in weeks 2 and 4 on glass tiles, and weeks 4 and 7 on ceramic. Generally, the diversity index and evenness considerably varied on ceramic without any specific pattern. Photosynthetic component biomass growth rates on glass and ceramic substrates (Fig. 4a, b) and the diversity indices on these substrates did not show any correlation. The exponential growth phase on glass tiles was apparently characterized by a decrease, both in the diversity and evenness in week 2, pointing to the prevalence and dominance of specific colonizers that arrived in week 1, while the diversity equilibrium recovered in week 3. In the later period of colonization, the evenness of the community on the glass substrate was more or less stable, and the diversity showed a positive trend from the 4th week till the end of the study period. The increase in the diversity during the colonization time is often observed in studies where glass slides are used as an artificial substrate for the development of periphyton (Kralj et al. 2006; Bordoqoui, Ferragut 2012) and is usually explained by the constant arrival of new colonizers. On the other hand, when wooden substrates are considered (Hillebrand, Sommer 2000; Sabater et al. 1998.), the negative effect of the colonization time on the diversity is observed and theoretically explained by the competitive advantage of specific taxa in the later successional stages (Hillebrand, Sommer 2000). In our study, the periphyton biofilms developed on each substrate had one feature in common and this was the diversity decline in week 4 that occurred after the loss phase.
All algal taxa detected in quantitative analyzes were allocated to the ecological groups and the percentage contribution of groups, based on the number of ind. cm-2, is presented in Fig 6. Diatoms were grouped into the low profile, high profile and motile guilds according to Passy (2007), Passy & Larson (2011), Rimet & Bouchez (2011) and Gottschalk & Kahlert (2012). Most of the low profile guild members were representatives of
Percentage contribution of the ecological groups to the periphyton community during the duration of the experiment on glass (a), ceramic (b), willow (c) and yew (d) tilesFigure 6
When the percentage contribution of groups during the exponential growth phase (the first three weeks) is considered, our results showed specific patterns on each substrate. Communities developed on yew and willow tiles looked alike in this particular period (the first three weeks, Fig. 6b, c, d), although the ratios of the represented groups were different. During the exponential phase of growth, the unicellular and colonial group (immigrants from phytoplankton) clearly dominated on yew tiles, and this is also characteristic of the biofilm developed on willow. The motile guild was clearly represented only on ceramic tiles during the 1st week of colonization. In weeks 2 and 3, the percentage of ecological groups on ceramic tiles was generally stable, with only motile and high profile guilds underrepresented, but this trend was observed on all substrates. The glass substrate was clearly different during the exponential phase, which was also reflected in the diversity index (Fig. 5a), and Figure 6a shows that the major reason for the diversity decline in week 2 is the dominance of the low profile guild’s representatives. At the end of the exponential phase (week 3), the balance of the community on glass tiles was reestablished (H increased), however, still with the highest contribution of the low profile guild. Thus, it can be concluded that glass was preferred by low profile diatoms during the exponential growth phase, wooden substrates by the unicellular and colonial group, while ceramic was more or less equally colonized by soft bodied algae and low profile diatoms as well as the motile guild, but only at the very beginning of the exponential phase. Representatives of the low profile guild appeared to be pioneers in the colonization process, particularly on the glass substrate, while the unicellular and colonial group play the same role on the wooden substrates. Mihaljević & Pfeiffer (2012) reported that planktonic Cyanobacteria dominated in the periphytic community developing on glass slides in the very early colonization phase (day 1 of colonization), due to their abilities to produce mucilage and adhere to the substrate, but they were very quickly outcompeted by short-generation time diatoms (day 3 of colonization). A similar pattern can be observed in our results (Fig. 6a), where the significant contribution of the unicellular and colonial group (in this period mostly represented by
The fluctuation phase (from week 4 onward) was marked by a general increase in the percentage of the low profile guild on wooden substrates. This could indicate the postponed colonization of wooden substrates by low profile guild’s diatoms compared to inert substrates (glass and ceramic). The contribution of low profile guild’s representatives to the community increased during the fluctuation phase along with the losses in biomass (negative growth rate) and the low biovolume of these taxa is a reasonable explanation of this (how it seems) paradox. Once again, this confirms the importance of the low profile guild in the recolonization process, as it appears that the observed losses in biomass were the early stages of the recolonization process.
Interactive forward selection revealed that total phosphorus (TP), nitrates (NO3-), oxygen saturation (O2) and the permanganate index (PI) were statistically significant environmental variables (Fig. 7).O2 and NO3- were positively correlated with the first axis (r = 0.6945 and r = 0.6279, respectively), while TP showed negative correlation (r = −0.4683). PI was positively correlated with the second RDA axis (r = 0.5699). The first RDA axis explained the main portion of the total variance in our data (60.49%). Thus, the first axis represents the variation in the community explained by O2, NO3- and TP, and the second vertical axis represents the part of the variation explained by PI. The statistical analysis showed high significance (F = 14.6,
Relationship between the ecological groups of periphytic algae and environmental parameters Ecological groups: low profile guild (LPG), high profile guild (HPG), motile guild (MPG), unicellular and colonial (OCC), filamentous Cyanobacteria (CF) and filamentous Chlorophyta (CHF); Environmental parameters: total phosphorus (TP), nitrates (NO3 −), oxygen saturation (O2) and permanganate index (PI). Weeks of sampling (W1-W8) and substrates (Ceramic, Glass, Yew and Willow) are included as supplementary variables.Figure 7
All groups were positively correlated with TP, but only MPG was correlated with both TP and NO3- and unlike other groups, it was found in larger numbers on glass and ceramic compared to wooden substrates (Fig. 7)
Zhang et al. (2013) suggested that wooden substrates in general are a source of nutrients in available forms for the development of algae in biofilm, primarily due to the decomposition processes. The representatives of the motile guild are generally referred to as superior competitors in nutrient-rich habitats, because of their physical capability to select a suitable position in the biofilm (Passy 2007). This guild was underrepresented in our study and was mostly found only on glass and ceramic substrates (Fig. 6a, b). Compared to the wooden substrates, glass and ceramic could be considered inert, and this may be the reason why the motile guild occurred only on these substrates. The observed presence of the motile guild only on inert and not wooden substrates could indicate that the communities on wooden substrates were not limited by the supply of nutrients (primarily NO3-), because of the endogenous loading from the decomposition processes. Therefore, the motile guild could not exhibit the competitive advantage and proliferate on wooden substrates in a greater or more apparent portion. Based on our results and consistent with the suggestion by Stenger-Kovács et al. (2013), it can be concluded that the community developed on inert substrates (glass and ceramic) could provide more realistic insight into complex environmental changes.
According to the Lakatos index, communities developed on four kinds of substrates were very alike; biomass was mainly low and biofilm was usually inorganic and heterotrophic. When the autotrophic biomass (Chl
The dynamics of the diversity changes during the colonization on both wooden substrates was very similar and different in the communities developed on glass and ceramic. The low profile guild appeared to be represented by pioneers in the colonization process, particularly on the glass substrate, while the unicellular and colonial group played the same role on the wooden substrates. The fluctuation phase (from week 4 onward) was marked by a general increase in the contribution of the low profile guild on wooden substrates. Generally, the occurrence of the low profile guild’s representatives negatively affected the diversity and evenness of the periphytic community developed on glass, and evenness on ceramic and yew tiles, indicating the competitive advantage of taxa from the low profile guild in the recolonization phases. Nonetheless, this guild was not observed on the willow substrate in the recolonisation phase, instead the filamentous Cyanobacteria group occurred there. Successional patterns on each type of substrate appeared to be very specific.
The community developed on wooden substrates showed lesser sensitivity to organic compound loads compared to the inert substrates, which could be a consequence of the habitat preferences by specific guilds. The presence of the motile guild only on inert and not wooden substrates could indicate that the communites on wooden substrates were not limited by the supply of nutrients (primarily NO3-), because of the endogenous loading from the decomposition processes. Therefore, the motile guild could not exhibit the competitive advantage and proliferate on wooden substrates in a greater or more apparent quantity. It can be concluded that the community developed on inert substrates (glass and ceramic) could provide more realistic insight into complex environmental changes.