Planktonic algae abundance and diversity are similar in urban stormwater ponds of different geographic locations and natural shallow lakes

Water samples were collected from 5 locations distributed around the shore line of each pond and lake, approx. 20 cm below the water surface. The distance from the shore line for sampling was selected where the water depth was approx. 70 cm. Sampling locations are exemplified for ponds PDK1, PCAN1, and lake LDK1 in Fig. 1.

Samples from the 5 locations were pooled and mixed well for each water body. A subsample was preserved with Lugol’s solution for later phytoplankton identification and enumeration. The water quality parameters: conductivity (μS cm⁻¹), temperature (°C) and dissolved oxygen (mg L⁻¹), were measured on site at each of the 5 sampling locations with portable multimeters: WTW Multi 3430 set G (DK) and YSI-556 MPS (CAN). pH was measured on site as well, but only for Danish water bodies.

The pooled water samples were also analyzed for chlorophyll-a (μg L⁻¹) and total phosphorus (TP, μg L⁻¹) both in Canadian and Danish water bodies. For Canadian ponds, procedures described by Song et al. (2013) and McEnroe et al. (2013) were followed. For Danish water bodies, TP was measured according to a method adapted from DS 292 (1985). For chlorophyll-a extraction, water samples were filtered through 0.7 μm GF/F filters, and a modified 90% acetone method was applied (adapted from Bellinger and Sigee 2010). Briefly, filters were homogenized in acetone, kept in dark for 20 min, centrifuged at 2500 rpm for 10 min and chlorophyll-a measured using a spectrophotometer and absorption at 750 nm and 664 nm wavelengths. Water quality parameters, such as suspended solids, NO₃ + NO₂-N and dissolved reactive phosphorus (DRP) were measured only for Danish water bodies. Water samples for DRP and NO₃ + NO₂-N measurements were filtered through 0.7 μm filters. DRP and NO₃ + NO₂-N were analyzed according to a method adapted from Standard Methods 4500-P (SM 1999) and according to DS 223 (1975) respectively. Total dissolved nitrogen (TDN), seston nitrogen (SN), total dissolved phosphorus (TDP) and seston phosphorus (SP) were measured for Canadian ponds only, as described in Song et al. (2013) and McEnroe et al. (2013). Parameters measured in Danish water bodies only or Canadian water bodies only were not included in the statistical analyses.

Phytoplankton identification and enumeration was performed with an inverted microscopes (GX Optical XDS-3 and Motic AE31). Samples were analyzed using HydroBios or KC-Denmark counting chambers. Depending on sample richness, either 5 or 10 mL chambers were used. When necessary, corresponding sample dilutions were made with demineralized water. Identification was attempted at genus level. In cases where this was not possible, organisms were assigned to other taxonomic levels, such as family, order, class, phylum, or to a group of unidentified organisms. Filamentous green algae were not included in the identification and counting. Counting was carried out either for a whole chamber at 100x magnification in order to capture the largest organisms, or in transects at 400x magnification when smaller organisms were counted. 600x magnification was used for identifications or counting of the smallest organisms. For some samples a somewhat uneven distribution was unavoidable, which was compensated by doing the counts in transects. Throughout, a single cell, colony, or filament was considered as a counting unit. Counting was continued until 100 counting units of each taxa/group was reached, but not in more than 7 transects. Organisms which did not reach a 100, but significantly contributed to the total biovolume, were taken into account as well. 95% of a sample (by biovolume) was counted, taking into account the most abundant taxa and taxa significantly contributing to the biovolume. An approximation of this contribution was done by counting all organisms in one transect, estimating their biovolume and that way distinguishing the significant sample taxa. Biovolume was estimated for every taxa or group of organisms assigning geometric formulas, as described in Hillebrand et al. (1999) and Lauridsen et al. (2005). For organism identification, John et al. (2011) was used as the reference key. Other identification keys by SPIPHES (1982), Tikkanen et al. (1992), Kristiansen and Nygaard (2001), Wehr et al. (2003), Cronberg and Annadotter (2006), Komarek and Anagnostidis (2007), Komarek (2008, 2013), and Guiry (2017) were used when additional specifications were necessary to identify a certain organism.

Statistical data analysis was carried out using Past v. 3.15 (Hammer et al. 2001; Legendre and Legendre 2012) and Sigmaplot v. 12.3 (Conover 1980; Snedecor and Cochran 1989; Systat Software 2016). One-way ANOVA or one-way ANOVA on ranks (depending on data normality) tests were performed on the number of taxa, total number of organisms as well as total biovolumes of the three types of water bodies – Danish ponds, Danish lakes and Canadian ponds using Sigmaplot v 12.3. Algal community similarity among groups of ponds and lakes were tested using an analysis of variance on similarities (ANOSIM) between individual water bodies within the groups (Clarke and Warwick 1994). These analyses were performed in the Primer Software Package (Primer-E Ltd. 2016). The different water bodies were also compared by classical Bray-Curtis Cluster analysis and DCA analysis, intending to determine community and taxa similarity patterns among the ponds or lakes grouped together depending on their phytoplankton community composition using Past v. 3.15. The tests were performed using log-transformed relative abundance and biovolume data. Relationships among phytoplankton composition and water body related physical and chemical parameters were evaluated by Spearman’s rank correlations in Past v 3.15. Phytoplankton community and water quality data sets were also submitted to CCA analysis in Past v. 4 in order to test whether there was any urban gradient effect on phytoplankton community structure. Lake and pond water quality indicators were also submitted to a PCA test in Past v 4 in order to determine which ones were the major drivers of the differences between ponds and lakes.

Water quality parameters measured during the sampling period varied between Danish and Canadian water bodies (Table 2).

The measured conductivity varied over the season in the ponds, but not the lakes. It is likely related with runoff salt content due to road deicing applied during the cold season, although presence of other dissolved ions could have contributed to the measured values as well. With regard to nutrients, total phosphorus concentrations in Danish lakes were more similar to Canadian ponds than to the Danish ponds. Danish ponds had significantly (Tukey test, p < 0.05) higher total phosphorus concentrations (48–230 μg L⁻¹), compared to the other water body types. Also, chlorophyll-a concentrations were significantly (Tukey test, p < 0.05) higher in Danish ponds compared with Canadian ponds. In general, all concentrations increased during the summer and beginning of autumn, however, less markedly in Canadian ponds. Other parameters varied depending on the water body or sampling month and no significant differences were found among the three water body types, except for total suspended solids with significantly higher (p < 0.05) concentrations measured in Danish ponds compared with lakes.

A multivariate PCA analysis performed with pond and lake water quality indicators showed that ponds and lakes were quite different with respect to the measured conductivity and their catchment area. Such result is highly expected due to typically received higher runoff salt content by ponds and larger catchment areas of natural lakes.

The identified phytoplankton taxa were ascribed to 9 phyla – Chlorophyta, Cyanophyta, Euglenophyta, Cryptophyta, Dinophyta, Bacillariophyta, Chrysophyta, Xantophyta, and Haptophyta. In the whole data set a total of 101 taxa and specified groups of phytoplankton were observed: 14 to 57 in Danish ponds, 18 to 47 in Canadian ponds and 14 to 30 in Danish lakes (Table S1, supplementary material).

The majority of the phytoplankton taxa were present in all three types of the water bodies, such as, single-celled Chroococcales, desmids Closterium, Cosmarium and Stauraustrum of the order Zygnematales, Pseudanabaena filaments, cryptophytes, or pennate diatoms. Comparing ponds and lakes, several taxa or taxonomic groups of phytoplankton were more often observed both in Danish and Canadian ponds than in the Danish lakes. These included the green algae Ankistrodesmus, Acutodesmus, Desmodesmus, Monoraphidium, Pediastrum, Scenedesmus, Crucigeniella, Dictyosphaerium, all euglenophytes, and most of the chrysophytes, centric diatoms, and colonial or filamentous diatoms.

With respect to the geographic locations of the water bodies, three organisms from the green algae order Volvocales – Eudorina, Gonium, and Pandorina – were only found in Canadian ponds and only observed a few times in Danish lakes. Also cysts of euglenophytes, ascribed to a group “cysts”, were observed in one of the Danish ponds, PDK2, and one of the Danish lakes, LDK1, while they were abundant in the Canadian ponds.

One fifth of the identified taxa was ascribed to the phylum Cyanophyta, which comprises many species capable of producing toxins that may cause adverse effects on human health. Several of the identified cyanobacteria genera, namely Anabaena, Anabaenopsis, Lyngbya, Microcystis Oscillatoria, and Planktothrix are listed as some of the potential toxin-producing organisms with most significant impacts on human health (Chorus and Bartram 1999; WHO 2003). The presence of these genera varied during the sampling season. A slight difference was found between the analyzed ponds and lakes: in Danish ponds 0–4 of these genera were identified, in Canadian ponds 0–3, and in Danish lakes 1–2.

The highest numbers were counted for single-celled coccoidal and rod-shaped Chroococcales (Fig. S1, supplementary material), widespread picoplanktonic Cyanophyta, which are common in the plankton of ponds and lakes (John et al. 2011). In this study, they were dominating numbers of organisms in all samples from all water bodies. Several peaks of single cells from colonial organisms of Chlorophyta or Cyanophyta were observed in LDK3-M, LDK1-J, LDK1-S, PDK3-J, and PDK3-S. The total numbers of organisms showed a high variability among all water bodies and sampling months: from 5.7·10² organisms mL⁻¹ to 5.5·10⁵ organisms mL⁻¹ for Canadian ponds, 1.9·10⁴–4.1·10⁵ organisms mL⁻¹ for Danish ponds and 2.9·10³–1.5·10⁵ organisms mL⁻¹ for Danish lakes.

The estimated total biovolume showed a high variability among the ponds and lakes (Fig. 2). The biovolume in the Danish lakes was found to be dominated by cyanophytes (up to 96%) and chlorophytes (up to 43%). Also, several peaks of cryptophytes were observed in LDK1 and LDK2 in May or dinoflagellates in LDK1 in July and September. In some of the months, Danish ponds were dominated by cryptophytes (up to 69%), cyanophytes (up to 61%), euglenophytes (up to 78%) and chlorophytes (up to 80%), while in Canadian ponds a larger part of the biovolume (10% - 15%) was composed of diatoms (PCAN1-M-J, PCAN2-M, PCAN3-M) or chrysophytes (PCAN1-M-J, 35% and 9% respectively). An apparent contribution of euglenophyte cysts to the biovolume was also observed in Canadian ponds PCAN1-J, PCAN3-J, PCAN2-S, 32% – 39% and 76%, respectively.

With respect to the potential toxin-producing genera, blooms of Anabaena were observed in all Danish lakes in July and/or September. The genus contributed 54% - 90% to the total biovolume. Microcystis colonies were also observed in LDK1-J-S, but to a smaller extent – up to 2%. Although a slightly larger variety of these genera was present in Danish ponds, only PDK2 had a notable contribution to the phytoplankton biovolume by Anabaena (PDK2-M) and Microcystis (PDK2-S), 46% and 26% respectively. In Canadian ponds, potentially toxic genera (Microcystis, Oscillatoria, Planktothrix) comprised up to 7% of the total biovolume, with an exception of PCAN3-S, where 89% of the biovolume were Microcystis colonies.

Total taxa numbers, total numbers of counted organisms and estimated total biovolumes in Danish lakes, Danish ponds and Canadian ponds for the three sampling months were not different between water bodies (ANOVA, p > 0.05, F ≠ 1). Similarities in algal communities among the three groups of water bodies for each sampling month were also tested using ANOSIM tests. Similar to the ANOVA tests, the ANOSIM test did not reveal any significant differences among the three types of the water bodies, neither with respect to number of organisms or biovolumes (ANOSIM, p_org = 0.072; p_biovol = 0.077).

The multivariate DCA test did not show distinct grouping of the three types of water bodies either (Fig. 3; Fig. S2, supplementary material). Only with respect to the DCA on the estimated biovolumes (Fig. 3), the two axes of the respective plot were found to explain 26% and 17% of the pond and lake distribution and have significant correlations with several pond and water chemistry parameters. Axis 1 was found to significantly correlate with the surface area of the water bodies (r_s = −0.54; p = 0.004), and mean water depth (r_s = −0.42; p = 0.03), while axis 2 correlated with chlorophyll-a concentration (r_s = 0.5; p = 0.008) and temperature (r_s = 0.48; p = 0.01). To some extent, lakes were different from ponds, evidenced by their position on the lower part of axis 1 (Fig. 3). However, the observed differences are rather small, and it cannot be excluded that the observed differences between ponds and lakes are due to random variations.

A somewhat clearer result was obtained by CCA analyses (Fig. 4). Both when performed with community abundance and biovolume data, it could be observed that lakes grouped more distinctly from ponds, despite a few outliers. In Fig. 4 it can be seen that conductivity is one of the most important water quality indicators deciding the separation between the investigated ponds and lakes. Regarding the community composition, the main difference between ponds and lakes was a larger part of biovolume taken up by phyla Cyanophyta in lakes.