A new charophyte habitat with a stabilized good ecological potential of mine water

Each newly-created pond which is supplied with mine water gives the opportunity to study a unique ecosystem in context of possible conditions for biotic live. Therefore, this research aimed to assess a phytoplankton-based ecological potential against the trophic conditions and the risk of contamination with trace elements, and demonstrate the possibility to stabilize at least good water quality of a clarification pond. The gradual decrease in turbidity-related variables (including suspended solids and iron) and nutrients, on the one hand, and an increase in phytoplankton-related indicators, on the other hand, were the most evident. Besides, relative stability in trace elements (the best water quality class), trophic level (slightly eutrophic level) and ecological potential (maximum potential), and relative instability in sulfates and calcium were also recorded. The final stabilization of water habitat resulted in abundant growth of charophyte Nitella mucronata. This all suggested a new ecological opportunity for settlement of a rare species and important for biodiversity enhancement. Furthermore, the study revealed that a clarification pond did not pose any toxic risk from the elevated content of trace elements or the growth of toxic or potentially toxic cyanobacteria which is essential for proper functioning and management of water ecosystems.

it suggested a real possibility to establish the biological potential of such clarification ponds. The water bodies impacted by abandoned coal mines has recently become an interest of Environmental Agency in England 14 due to the implementation of the Water Framework Directive (WFD) but mainly in the context of mine water discharges and the possible pollution.
The aims of the study were to (1) assess a phytoplankton-based ecological potential against the trophic conditions and contamination with trace elements, and (2) demonstrate the possibility to stabilize at least good water quality of a newly-formed clarification pond for a new habitat during the 6-year period. This research can be treated as an approach to further manage of the water resources including the newly formed water bodies not only in local or European scale but also in global scale regarding freshwater management, policy and conservation.

Results
Environmental variables, toxic risk and trophic conditions. During the 6-year period, the highest average temperature and oxygen saturation were noted in summer, while their lowest averages in winter (Supplementary Table S1). In summer, temperature ranged from 18.1 to 21.7 °C and oxygen content from 8.1 to 9.8 mg L −1 . In winter, in turn, temperature and oxygen content ranged from 9.7 to 13.1 °C and from 8.6 to 11.8 mg L −1 respectively. Both the spring and autumn seasons were characterized by similar averages of water temperature and oxygen content. The thermal-oxygen conditions were not statistically different between the years (P > 0.05).
The water in the Kuźnica clarification pond was slightly alkaline with pH range of 7.4-8.4 (Supplementary Table S2). The SDD ranged from 0.42 to 0.69 m, on average, and it was statistically lower in 2014 than in 2018 and 2019 (P = 0.0003). Significant differences were observed in turbidity between two periods (P = 0.0003) with averages of 37.  Table S3). Their highest concentrations were as follows: 16.78 mg L −1 (in 2016), 3.84 mg L −1 (in 2014) and 0.36 mg L −1 (in 2017), respectively. Statistically significant differences were noted only for Fe (P = 0.0028). The concentrations of other trace elements were characterized by moderate variability (Table 1). Among them, the highest ranges of concentration were for Al (1.14-2.97 µg L −1 ), Pb (0.42-1.06 µg L −1 ), Cu (1.52-2.42 µg L −1 ), Ni (2.01-3.01 µg L −1 ) and Zn (20.01-33.96 µg L −1 ). The average concentrations of Ag, As, Cd and Hg, were about 0.01 µg L −1 in most samples, while for Se it did not exceed this value. Thus, their content corresponded well to the best water quality class. Summing up, no toxic risk from the elevated content of trace elements was also noted.
The partial indices of TLI were markedly differentiated ( Fig. 1), with the highest values of 4.9-6.5 and 5.4-6.0, respectively for TLI TP and TLI SDD . They covered the classes from eutrophic to hypertrophic levels. The TLI TN values varied (3.6-3.9) within the class characteristic for mesotrophic waters. The lowest values (1.7-2.5) and typical of microtrophic and oligotrophic levels were recorded for TLI Chl . Thus, the average values of partial indices pointed to levels from supertrophic to oligotrophic and confirming the general relationships as follows: The total phytoplankton density gradually increased from 0.40 × 10 5 ind. L −1 in 2014 to 20.78 × 10 5 ind. L −1 in 2019, on average ( Fig. 2A). Diatoms (19-76% of the total density), chlorophytes (14-74%) and cryptophytes (3-44%) dominated the phytoplankton. The total biomass ranged then from 0.05 to 1.39 mg L −1 , with seasonal averages of 0.05-1.14 mg L −1 (Fig. 2B). Diatoms, representing 41-67% of the total biomass dominated the phytoplankton assemblages throughout the almost whole study period. The exception was in 2015 when small chlorophytes formed the highest biomass (61% of the total biomass), and dinoflagellates and diatoms about 19% and 14%, respectively. Besides diatom dominance, euglenoids and cryptophytes had a share of 21% and 7%, respectively in 2014. In 2016-2018, in turn, dinoflagellates had 10-34%, cryptophytes 8-25% and chlorophytes 5-24%, and in 2019 only cryptophytes (with 35%) co-dominated with diatoms. Simultaneously with gradually increase in phytoplankton density and biomass, the chlorophyll a content also increased from 0.7 to 2.4 µg L −1 , on average in a temporal scale (Fig. 2C). However, during the growth season it changed in a generally narrow range, from 0.4 to 4.5 µg L −1 .
The most numerous were the small-sized species, i.e. diatoms: Cyclotella meneghiniana Kützing, Nitzschia palea  (Table 2A). They represent the coda of different habitat-related functional groups: C, D, X2, X1, F and Y. The dominance structure based on biomass was a slightly different. However, the highest biomass formed also the above mentioned species of the genera Chlamydomonas and Cyclotella (Table 2B) Table S4). The final multimetric PMPL indicated, thus, a maximum ecological potential throughout the whole study period. It was consistent to relative small phytoplankton biomass and chlorophyll a content, and their insignificant fluctuations did not affect any changes in the ecological classification.  Fig. S1) was identified for the first time in an early spring of 2019. After that, it began to grow rapidly to the end of the growth season. In spring, the biomass of N. mucronata was slightly diversified between different sites and it ranged from 26.58 to 32.60 g DW m −2 in the inflow and outflow sub-zones, respectively (Table 3). A seasonal increase of the average biomass was found in each zone of the pond. The more pronounced increase was observed in the outflow zone. There was recorded a significant increase of N. mucronata biomass (P < 0.01), from 32.60 to 323.39 g DW m −2 in spring and autumn, respectively. Only, in the inflow sub-zone the increase of the biomass (26.58 g DW m −2 , 43.71 g DW m −2 and 152.35 g DW m −2 in spring, summer and autumn, respectively) was statistically insignificant. Generally, a statistically significant increase of the average biomass of the charophyte from 29.01 to 246.95 g DW m −2 (P < 0.001) was recorded throughout the whole growth season.
Sequential ecosystem changes. Studying the 6-year-long sequential ecosystem changes and settlement potential, it can be summarized as follows (Fig. 4): ) and habitat template of coda, C eutrophic small-and medium sized lakes with species sensitive to the onset of stratification, D shallow turbid waters including rivers, F clear, deeply mixed meso-eutrophic lakes, X1 shallow, eu hypertrophic environments, X2 shallow, meso-eutrophic environments, Y this codon, mostly including large cryptomonads but also small dinoflagellates, refers to a wide range of habitats, which reflect the ability of its representative species to live in almost all lentic ecosystems when grazing pressure is low, MP frequently stirred up, inorganically turbid shallow lakes, W2 meso-eutrophic ponds, even temporary, shallow lakes, L O deep and shallow, oligo to eutrophic, medium to large lakes, U stratifying oligotrophic and mesotrophic lakes, where nutrient resources are exhausted in the upper layers but still available in the darker deep ones, the essential adaptation under these conditions is the combination of motility with large size, -not observed. Trachelomonas armata Scientific Reports | (2021) 11:14564 | https://doi.org/10.1038/s41598-021-93827-z www.nature.com/scientificreports/ 1. gradual decrease in turbidity, electrical conductivity, suspended solids, carbon, phosphorus, nitrogen, iron, and some ions; 2. gradual increase in chlorophyll a, phytoplankton indicators (such as: density, biomass, richness and biodiversity) and water transparency; 3. stability in temperature, oxygen, pH, bicarbonate, potassium, most of the trace elements, and trophic level and ecological potential; 4. relative instability in sulfates and calcium; 5. final stabilization of water habitat and a new ecological opportunity for the settlement of charophyte Nitella mucronata.
The processes that have accompanied the ecosystem changes included primarily the effective sedimentation, nutrient-iron bounding and their release to the bottom, decreased bioavailability for primary producers. In consequence, an effective limitation of the phytoplankton growth can successfully maintain the maximum ecological potential on the long-term scale.

Discussion
The Kuźnica clarification pond gives the opportunity to study a unique water body concerning the possible biotic live. This pond was selected for the studies on the possibility to colonize by primary producers, then, other organisms with an appearance of new species, and consequently the changes in an ecological potential against the variability of physical and chemical parameters of the mine water. The relatively low TDS content (not exceeding 529 mg L −1 ) and slightly increased conductivity, which should be associated with the increased content of sulphates, carbonates and chlorides, proved the origin of the freshwater supplied to the clarification   www.nature.com/scientificreports/ pond. This state reflects "specific" geochemical conditions, and a high concentration of calcium and carbonates stabilized the slightly alkaline pH of the water in the Kuźnica pond. According to Gogacz 15  The above mentioned changes could be directly related to the progressive exploitation of lignite deposits. As a result, the exposure to transitional rocks within the Mesozoic-neogen contact zone has then increased. According to Pękala 16 , these rocks consist mainly of silica and calcium oxide as well as calcite with variable iron and manganese content. In good oxygen conditions, iron and manganese significantly influenced the turbidity and water transparency in the Kuźnica pond. Suspended solids, which are characteristic of the aquatic environment of the clarification pond, were, thus, the transporter for the precipitate of iron and manganese hydroxides. Similar results were described by other authors 17,18 .
The variability of geochemical phenomena in the Bełchatów mine was favored by the local tectonics of the mesozoic substrate, and the presence of halite diapir located between two exploited openings 16,19 . The research also showed that sodium chloride anomalies (effect of water ascension) and manganese-iron anomalies were noted in some wells of the drainage system. The hydrochemistry of the Kuźnica clarification pond and the water quality in the surface drainage system were also influenced by other factors related to the progress of the www.nature.com/scientificreports/ exploitation front. Then, the new layers of sediments were exposed and their structure was loosened. The progresses of the operating front and controlled shutting down of the drainage wells resulted in the groundwater restoration 19 . Additionally, the reconstruction of the groundwater level is constantly renewed around the Szczerców opencast mine. This process was initiated in 2012, i.e. before the construction of the Kuźnica pond. At that time, the formation of an internal heap began, and the rebuilding rate of the groundwater level depended on its precipitation infiltration. Thus, the observed changes and stabilization of the chemical composition of water in the Kuźnica pond were directly influenced by many interrelated factors. In Poland and especially in the area of studied water body, there was not recorded any elevated content of heavy metals. In current studies, the relatively low content of studied trace elements allows the Kuźnica clarification pond to be classified as waters having at least the second quality class, i.e. good ecological potential in accordance with the Polish Regulation 20 . There were no cases of exceeding the permissible concentration limits of trace elements. Generally, the mining activities have become the historically and globally important contributors to heavy metal pollution of many water bodies. In Poland, and especially in the area of mining activities, such situation with a high content of heavy metals was not recorded yet 16,21,22 .
The newly-formed clarification pond studied here was generally characterized by very low phytoplankton growth and diversity similarly to the findings recorded in the other newly-created post-mining lakes in Poland 22 . However, the 6-year-long ecosystem changes were related to a very slow but gradual increase in chlorophyll a content and phytoplankton features, i.e. density, biomass, richness and biodiversity. Current study, and just like previous study on the possibility of using this pond to extensive aquaculture 13 , suggest that phytoplankton could be limited by a large amount of suspended solids during both the mechanical and shading processes. These include primarily the sinking of phytodetrital aggregates and a light limitation 23,24 , as well as negative relations with zooplankton. The negative relationship between phytoplankton features (biomass, density, chlorophyll a) and environmental variables (total phosphorus, ammonium, TSS, ISS, TDS, iron and EC) was also confirmed. Simultaneously, the positively-related to phytoplankton water transparency tended to gradually increase, and it was related rather to the decrease of suspended solids content but not to phytoplankton growth. A phenomenon of reduced visibility in the water column by organic matter was also characteristic of the post-mining lakes 25 .
Low values of total biomass, cyanobacterial biomass and content of chlorophyll a in the whole study period of 2014-2019 were typical of high water quality, i.e. maximum ecological potential of the Kuźnica clarification pond in accordance with the ecological classification of European water bodies 7 , and close to the unimpaired state, i.e. reference conditions common for European lakes 26 . The biodiversity Shannon index pointed at moderately or slightly polluted waters based on classifications given in Napiórkowska-Krzebietke 27 , whereas the final TLI indicate a slightly eutrophic level. Based on functional classification 28,29 , the dominant species of are associated with different habitats. The most abundant diatoms are characteristic for eutrophic, shallow, turbid (primarily inorganically) water bodies, next, the small-sized green algae are typical for shallow, meso-eu-hypertrophic environments or even clear, deeply mixed meso-eutrophic lakes similarly to cryptomonads which refer to a wide range of habitats. The large-sized dinoflagellates, i.e. Ceratium hirundinella and Peridinium cinctum prefer deep and shallow, from oligo to eutrophic ones. In opposite, the euglenoid Trachelomonas armata occurs primarily in meso-eutrophic small ponds, and the euglenophytes often exist in water containing organic pollution 30 .
Furthermore, in 2019 i.e. in the 6th year of phytosociological observations, the clarification pond was inhabited by N. mucronata. This charophyte is included as one of the rarest Polish charophytes and considered as critically endangered or extinct species on the Red list of algae [31][32][33] . Currently, N. mucronata is also found in shallow artificial water bodies, which are characterized by trophic poverty and good light conditions 33,34 . For the growth of charophytes, an increased concentration of calcium ions and a slightly alkaline water pH are very important 35,36 , and such conditions are met in the water of the Kuźnica pond. However, the occurrence of N. mucronata in the turbid environment has not yet been recorded. The most charophytes cannot grow in water that is permanently very turbid and enriched in nutrients, but some of them can photosynthesize at low light intensities, or can survive in some periods of turbid water 37 . Therefore, the abundant occurrence of N. mucronata's monospecies community in the Kuźnica clarification pond proves the high colonization potential for this species. Its maximum biomass recorded in autumn 2019 (nearly 247 g DW m −2 ) was relatively high and similar to the results from the other study 38 . This can prove the wide tolerance of N. mucronata for hydrochemical parameters and light availability. It is worth noting that Kuźnica pond was poor in nutrients, and enhanced water turbidity was not a result from the increased chlorophyll a content or phytoplankton biomass but iron hydroxides had a significant influence on water transparency and turbidity. The findings of Fontanini et al. 39 showed that N. mucronata is perfectly suited for environments with increased iron content. The research showed that not only the light availability, but also the factors which determine the penetration of light in the water can affect the expansion of the charophyte. Thus, the appearance of N. mucronata was not only the result of the improvement of light conditions but also of the favourable water chemistry. Similarly to the findings of Blindow et al. 9 , the rapid development of N. mucronata in the clarification pond was the result of the stabilization of environmental variables and the ecological potential. Such stabilization in the shallow Kuźnica pond was a result of separating the sediments from the water which highly limited their resuspension. The reducing of resuspension has been a key contribution of macrophytes to an improvement of the water transparency in shallow water bodies 40 . The ability to act as a nutrient sink is also a stabilization factor which can cause the decrease in the nutrient availability for phytoplankton and epiphyton 41 .
The 6-year-long sequential ecosystem changes and settlement potential confirmed that natural processes allowed the turbid water of the shallow pond to reach the phase of stabilized environmental conditions. The key role of Nitella mucronata in stabilizing the maximum ecological potential in a newly-formed clarification pond was also suggested. Such waters were characterized by environmental variables typical of natural lakes. In previous studies, a limited phytoplankton and zooplankton growth with good growth rate of omnivorous L. idus were confirmed 13 . Now, in this study, we indicate that environmental conditions of clarification pond can support also www.nature.com/scientificreports/ the proper functioning of ecosystem. The multi-seasonal results proved also that the pond fed with water from the opencast mine drainage system can be safely used for utility purposes, one of which is recreational fishing. All changes in trophic state or ecological status have an impact on the habitat conditions and the growth rate of many fish species, including those in artificial lakes 42,43 . Therefore, the stabilization of low trophic level and maximum ecological potential is an inspiration for further research on fish habitat in such ponds.

Conclusions
The 6-year-long research on ecosystem changes in the Kuźnica clarification pond confirmed a final stabilization in low nutrient enrichment and non-algal turbidity-related variables. Generally, the limited phytoplankton growth and especially low cyanobacteria biomass and chlorophyll a content but with a gradual and slight increase were recorded throughout the whole study period. The phytoplankton assemblages were dominated by species with different habitat requirements including meso-eu-hypertrophic turbid waters as well as oligotrophic ones. Diatoms of the genera Cyclotella, Nitzschia, Ulnaria and Staurosira, green algae of the genera Chlamydomonas, Monoraphidium and Kirchneriella, and cryptomonads of the genera Plagioselmis and Cryptomonas were dominants. The final phytoplankton-based assessment indicated a maximum ecological potential whereas TLI-based assessment indicated a slightly eutrophic level in the Kuźnica clarification pond. A low content of trace elements and low biomass of toxic or potentially toxic cyanobacteria demonstrated a very low toxic risk.
Our study confirmed the possibility to inhabit and abundantly grow of the sensitive charophyte considered as critically endangered or extinct species Nitella mucronata in water of clarification pond. N. mucronata played an important role in the stabilizing the environmental conditions and maintaining at least good ecological potential in a newly and formed clarification pond. Therefore, the clarification pond can support populations of charophyte species, and provide a new habitat for aquatic species that is less available in naturally occurring shallow and turbid water bodies of the region.

Materials and methods
Study area. The study site is a newly-formed pond created in 2013 to pre-treat water from dewatering system of the largest brown coal strip mine in Poland so-called "Bełchatów" (51° 13′ 29.5″ N, 19° 9′ 26.5″ E, Fig. 5A). Its basic role is a technical function in reducing the excess of suspended matter throughout the sedimentation process, thus, it can be called as clarification pond. The Kuźnica clarification pond receives the waters originating from different depths of the surface drainage system (surface and ground waters from shallow wells mixed in variable proportions) of the open pit in Szczerców. The pond is surrounded by stone embankments equipped ), and other selected ions (Mg 2+ , Cl − , Na + , K + ). All these analyses were conducted according to standard methods 44 . Furthermore, the content of selected trace elements, primarily: Fe, Mn, Si, Ag, Al, As, Cd, Cu, Hg, Ni, Pb, Se, Si, Zn was determined with the accuracy of 0.01 μg L −1 according to the methodology based on an elementary coupled plasma ionization mass spectrometry (ICP-MS) using an emission spectrometer Elan ICP-MS (Perkin Elmer). The trace elements' selection for the determinations was made based on the results of the hydrogeochemical background performed for the area covering the mining activities 16,21 . Such a range of hydro-analyses coincides with the cyclic monitoring of the mine water quality supervised by the Polish National Hydrogeological Service 15 .
Biological parameters and analyses. The integrated phytoplankton samples at 1 m depth were also taken out in the three-five times regime throughout the growth season, i.e. in spring, summer and autumn. The samples for taxonomic analysis were additionally taken using the plankton net with 10 μm mesh size. The quantitative analysis covering phytoplankton density and biomass was performed using an inverted microscope according to standard Utermöhl method 45 . The counting organisms (single cells, colonies or filaments) were examined at different magnifications: 100×, 200× and 400× for large-, medium-, and small-sized taxa, respectively. The qualitative analysis (taxonomic identification) was performed using a light microscope at magnifications of 200×, 400×, and 1000× with oil immersion. The total biomass and biomass of phytoplankton taxa were calculated on the basis of the cell biovolume measurements according to standard and revised method 46 . Taxonomic identifications were based on the latest references and verified according to currently accepted taxonomic names given in AlgaeBase 47 .
Phytosociological observations were also conducted from 2014 to 2019. In 2014-2018, no submerged macrophytes were found at the bottom of the main water zone. At the end of February 2019, charophyte Nitella mucronata (A.Braun) Miquel was observed for the first time and the expansion of this single-species Nitelletum mucronatae community was, thus, monitored. Taxonomic identification was made according to Pełechaty and Pukacz 48 . The observations were carried out on a total of 15 sites in the main water zone, including 5 sites each in the sub-zones: inflow (within the vicinity of the pre-sedimentation chamber), central and outflow (within the vicinity of the plant filter). Charophyte was collected regularly at a monthly interval throughout the growth season, i.e. spring, summer and autumn, using a bottom sampler according to Ekman-Birge with grasping area of 0.04 m −2 . Samples were cleaned of sediments, identified and dried in an air-forced circulation dryer at 70 °C until a constant weight was obtained. The biomass of submerged macrophyte was expressed in grams of dry weight (DW) per 1 m −2 of bottom area.
Functional, ecological and trophic classifications. The species richness expressed as the total number of phytoplankton species, and diversity indices, i.e. Shannon Index 49 and evenness 50 were chosen to describe phytoplankton biodiversity. The values of diversity indices were calculated based on species density. Determination of the main phytoplankton representatives of functional groups was based on the functional classification 28,29 .
Due to a lack of concern such a water body in Polish regulations but also across the European countries 51 , in the ecological potential assessment of the Kuźnica clarification pond the criteria were assumed as follows: • small, shallow, non-stratified, artificial water body, • catchment area has no impact, it could be the most similar to very low impact, • (Schindler's ratio, SR-ratio of catchment area and lake volume, SR = 0 ≈ SR < 2), • high Ca content > 25 mg dm −3 , • sampling regime: three-four times throughout the growth season, • the equations (1-5) for assessment were adapted Napiórkowska-Krzebietke et al. 52 , and references therein.
The Trophic Level Index (TLI) according to Burns et al. 53 was selected for trophic state determination. It comprises four partial indices based on total phosphorus (TP), total nitrogen (TN), Secchi disk depth (SDD) and chlorophyll a (Chl a). www.nature.com/scientificreports/ Statistical analyses. Non-parametric analysis of variance (Kruskal-Wallis test) was used to assess the differences between years concerning the physical and chemical parameters of the water and a biomass of submerged macrophyte in the clarification pond. All the analyses were performed with Statistica 13.0 for Windows, Statsoft, Tulsa. The level of significance was set to a P < 0.05. The relationships between phytoplankton characteristics and environmental parameters were tested with the redundancy analysis (RDA) of canonical ordination. The Monte Carlo permutation test (999) was then used. All tested parameters were standardized using log (x + 1)-transformation. Response data were compositional and had a gradient of 0.1 SD units long, thus, a linear method was recommended. The explanatory variables were additionally chosen after the analysis of variance inflation factor (VIF) and variables that had a VIF smaller than 10. The RDA was performed for phytoplankton characteristics (biomass, density and chlorophyll a) and nine environmental variables: EC, TDS, TSS, ISS, turbidity, SDD, ammonium, TP and Fe.
Ethical approval. The study on charophyte Nitella mucronata, which belongs to macroalgae, complies with local and national regulations. It was conducted outside the natural aquatic ecosystems and outside the habitats covered by forms of area protection. Samples were taken in artificial water body located in the area of active mining site. In cooperation with the PGE Mining and Conventional Power Generation SA Branch KWB Bełchatów, for collection of charophyte samples, all relevant permits and permissions have been obtained.