Introduction

Nutrient enrichment in lakes refers to the increase in nutrients, especially nitrogen and phosphorous due to anthropogenic activities that are responsible for the excessive growth of algae and aquatic macrophytes, thereby increasing the primary productivity of the lakes (Hampton et al. 2018). It disturbs the whole ecological balance of the lacustrine ecosystem by changing their trophic states (Elliott and Whitfield 2011; Qin et al. 2013; Dubey and Dutta 2020).

Aquatic macrophytes are important components that contribute to the primary productivity, stabilization, storage and cycling of nutrients within the lakes. Decline in submerged vegetation and increase in abundance of free-floating macrophytes are the most significant changes along the ecological gradient of the lakes (Jeppesen et al. 2012; Poikane et al. 2018; Dos Santos et al. 2020). Macrophytes are sensitive to eutrophication, water level fluctuations, acidification, shoreline modifications, recreation activities, navigation and biological invasion (Poikane et al. 2018). The growth and life cycle of free-floating macrophytes are mainly affected by the lake water chemistry, as they are in direct contact with lake water. Rooted submerged macrophytes are affected by both water and sediment chemistry as their bodies are inside the lake water and their roots derive nutrients from the benthic region sediments (Krems et al. 2013; Reitsema et al. 2018). Submerged macrophytes show better dermal absorption characteristics because of the structure of their leaves having underdeveloped lamina and skin, supporting the fast exchange of matter with the environment (Maleva and Nekrasova 2004). The emergent macrophytes are least affected in comparison to free-floating and submerged macrophytes by the water chemistry of lakes as they derive their major nutrients from the benthic sediments (Short et al. 2016).

Some aquatic macrophytes that inhabit polluted or nutrient-rich waterbodies absorb pollutants to maintain homeostasis in their internal cellular organization (Marecik et al. 2006). They develop effective defense mechanisms for maintaining homeostasis either by avoiding the uptake of metal ions from entering into them or by stopping or immobilizing the metal ions in the cell membrane and by the production and activation of various organic acids, amino acids, antioxidant particles and enzymes in their body (Mishra et al. 2006). Many aquatic macrophytes are known to have phytoremediation potential. They are tolerant to a wide range of pollutants including nitrogen, phosphorus and heavy metals as they have the ability to accumulate these pollutants into their cells, thus resulting in a high level of pollution removal (Ghosh and Singh 2005). Macrophytes are used in biomonitoring of the lacustrine ecosystems, due to their ability to tolerate and accumulate large amounts of pollutants—sometimes without damaging themselves and sometimes with anatomical and elemental changes. Biomonitoring studies of the lake ecosystem are mainly done using floating and submerged macrophytes as indicators (Kelly and Witton 1998: Ali et al. 1999; Krems et al. 2013).

Studies of macrophytic responses to eutrophication exposure on anatomical and elemental changes have been done mostly (Zhu et al. 2018) in the laboratory or controlled experimental conditions with fewer field investigations (Zhu 2012). Changes in anatomical structure along with elemental variations are key strategies against the stressed aquatic environment (Zhu et al. 2018). Consequently, the objective of this study was to investigate the change in anatomy as well as elemental constituents in common aquatic macrophytes from three different freshwater lakes having different trophic states. Anatomical and elemental traits were monitored using SEM and EDS. The changes on adaxial surface of leaves of Eichhornia crassipes and Hydrilla verticillata were examined using SEM, including stomata structure, number and surface morphology. Further, elemental changes in Eichhornia crassipes, Hydrilla verticillata and Typha latifolia were evaluated for three different lakes with different water quality, trophic state and catchment characteristics using EDS. This allowed to study how elemental constituents in the macrophytes increased with increase in trophic state. This is the first in-situ study reporting the anatomical and elemental responses of aquatic macrophytes against wastewater-induced nutrient enrichment in the freshwater tropical lakes of the Central Gangetic Plains. This study will be helpful in understanding the macrophytic response which shall be helpful in proper management of freshwater ecosystem.

Methodology

Study area

Three freshwater lakes have been selected with different catchment characteristics and pollution stress under the same climatic conditions in the Central Gangetic Plain of Uttar Pradesh, India. Lake 1 (Samaspur lake or L1) from the rural catchment of the Salon village in Raebareli district, Lake 2 (Kathauta lake or L2) and Lake 3 (Haibatmau lake or L3) from urban catchments of Lucknow district of North India were selected (Sampling sites images are provided in Fig. 1 of supplementary data). Water samples and macrophytes samples of selected free-floating, submerged and emergent macrophytes were collected from each of the selected lakes. A brief description of these three lakes is provided in Table 1.

Fig. 1
figure 1

Flowchart depicting study design and methodology

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Table 1 Brief descriptions of the three selected freshwater lakes
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Collection of water sample and water quality analysis

Physico-chemical parameters of the lake water were analyzed using standard sampling procedures (APHA 2017). Sampling was conducted in the month of October 2018. Fourteen water quality parameters including temperature, pH, secchi depth (SD), total dissolved solid (TDS), total suspended solids (TSS), electrical conductivity (EC), dissolve oxygen (DO), biochemical oxygen demand (BOD), alkalinity, hardness, nitrate, nitrite, total phosphorous (TP) and Chlorophyll a were analyzed for each of the three lakes (Lumb et al. 2011) (as given in Table 1 of supplementary data). Temp, pH, SD, TDS and DO were monitored onsite, whereas rests of the parameters were analyzed in the university laboratory. Water samples were collected from six sites of the selected lakes in triplets and the means along with standard deviation (SD) are provided in Table 2. A flowchart depicting study design and methodology is provided in Fig. 1.

Table 2 Water quality and trophic state of lakes during post-monsoon sampling
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Assessment of trophic state of selected lakes

The most commonly used classical method for the classification of lakes in different trophic states related to biomass is based on the productivity of lakes and is known as trophic state index (TSI). It is defined as the total weight of the biomass in a lake at a precise time and location (Carlson 1977). TSI is a valuable tool in identifying trophic state and productivity of the lacustrine ecosystem (Hillsborough 2008). It also indicates the overall health of the lacustrine ecosystem in terms of its nutrient status and the biomass it supports. This method uses lakes transparency (Secchi depth), total phosphorus and chlorophyll a for calculating their trophic state. The Transparency of the lake was measured using Secchi disk of diameter 20  cm and values are expressed in meters. Reading was taken at the maximum depth at which disk could be seen when lowered into the lake. Total phosphorous was analyzed by the method described in (APHA 2017) and chlorophyll a was estimated by the Acetone method using a spectrophotometer (Marker 1972). Chlorophyll was extracted in 80% acetone and the absorbance at 660 and 620 nm was recorded. The amount of chlorophyll was calculated using the absorption co-efficient. TSI values lie between 0 and 100, which categorizes lakes in different trophic states (Carlson and Simpson 1996).

The formulae used for calculating the Carlson trophic state index (TSI) for chlorophyll a, Secchi depth and total phosphorous (Carlson 1977) are mentioned below:

  1. 1.

    TSI (Chl-a) = 9.81ln Chlorophyll-a (µg/L) + 30.6

  2. 2.

    TSI (SD) = 60 – 14.41 ln Secchi depth (Meters)

  3. 3.

    TSI (TP) = 14.42 ln Total phosphorous (µg/l) + 4.15

Where Chl-a is (Chlorophyll a), SD is Secchi depth and TP is total phosphorous.

The overall Carlson Trophic State of the lakes was calculated using the modified Carlson and Simpson (1996) formula:

$${\text{TSI }}\left( {{\text{Overall}}} \right)\, = \,\left[ {{\text{TSI }}\left( {{\text{SD}}} \right)\, + \,{\text{TSI }}\left( {{\text{TP}}} \right)\, + \,{\text{TSI }}\left( {{\text{Chl}} - a} \right)} \right]/3$$

Collection of samples of macrophytes

Areas measuring 5 m × 5 m were selected in the selected three lakes from which free-floating Eichhornia crassipes and submerged Hydrilla verticillata were collected for morpho-anatomical and elemental profiling of the plant. Emergent macrophytes were collected from the littoral area of the lakes. After collection of the macrophytes, they were washed with distilled water to remove the attached debris and unwanted particles attached with leaves, stem and roots for further microscopy and SEM and EDS analysis.

SEM of macrophytes

For SEM analysis, macrophyte samples of 2–4 mm were washed in 0.1 M phosphate buffer three times each for 15 min at 4 °C to remove the unreactive fixative. The plant material was then dehydrated with 30, 50, 70, 90, 95 and 100% solutions of acetone. Each dehydration was carried out for 30 min at 4 °C (Pathan 2008). Plant samples were mounted onto the aluminum stubs and carbon tape. The samples were then coated with palladium using a sputter-coater to make them conductive. The specimens were observed using a scanning electron microscope (Model JSM-6490LV, Manufacturer Jeol Japan).

EDS of macrophytes

Eichhornia crassipes, Hydrilla verticillata and Typha latifolia were collected from the selected areas of 5 m × 5 m. Leaves, stems and roots of Eichhornia crassipes, leaves and stems of Hydrilla verticillata and leaves of Typha latifolia were washed with distilled water and samples were sun-dried for 15 days. Fine powders of the dried samples of the various vegetative parts of the macrophytes of different lakes were used for elemental analysis using EDS (OXFORD INCA X- act model 51-ADD0013 having resolution at 5.9 keV).

Statistical analysis

The means and standard deviations of the water quality parameters were calculated by using Microsoft Office Excel®. ANOVA and correlation analysis were performed using SPSS 20 software. Principal Component Analysis (PCA) and Cluster Analysis (CA) were performed using PAST 3.0 software (Hammer et al. 2001). CA employed Euclidean distance and non-weighted clustering using arithmetic mean (UPGMA) for various water quality parameters.

Results and discussion

Water quality of lakes and pollution stress

Water quality of the selected lakes was analyzed for various physico-chemical parameter such as Temp, pH, Secchi depth, TSS (Total Suspended Solids), EC (Electrical Conductivity), DO (Dissolved Oxygen), BOD (Biological Oxygen Demand), Alkalinity, Hardness, Nitrate, Nitrite, Chlorophyll a. The mean of water quality parameters of six sampling sites of Lake 1 (L1)], Lake 2 (L2) and Lake 3 (L3) is provided in Table 2. During the sampling period, Lake 2 was fragmented into two segments for cultivation of Trapa natans and fishing. In the fishing segment, the lake was found to have mesotrophic water quality and high algal growth. Lake 1 selected for the study is under the protection of the Uttar Pradesh Forest Department and within this lake point and non-point sources of pollution were regulated and monitored. Lake 1 was found to be oligotrophic, with a TSI value of 26.51, whereas Lake 2 and 3 were found to be in mesotrophic condition during the time of sampling, having TSI values of 47.57 and 43.09, respectively. The results of water quality analysis and trophic states along with ANOVA analysis of all the three lakes are provided in Table 2. The water quality parameters showed significant differences among the selected three lakes.

Principal component analysis (PCA) and cluster analysis (CA)

The result of analysis of the 14 physicochemical parameters was also subjected to multivariate exploratory principal component analysis (PCA). PCA showed that 99.99% of data variation is explained by two components. Component 1 accounts for 80.61% variation and Component 2 accounts for 19.38% variation. According to the rotated matrix component, Component 1 has main variables as Total Suspended Solids (TSS), BOD, Alkalinity, Hardness. According to the loading value of PCA, Component 2 was heavily influenced by TDS (Total Dissolved Solids) and Electrical Conductivity (EC) according to the loading value of PCA (Fig. 1 Supplementary file Table 1). The data show that the main vectors that influence component 1 are directly related to the degraded water quality of Lake 2 and Lake 3, which are in mesotrophic condition. High Electrical Conductivity (EC), Total dissolved solids (TDS), Hardness, Alkalinity, Total suspended solids (TSS) indicate the major effects of anthropogenic activities on Lake 2 and Lake 3 as shown in (Fig. 2). This finding confirms the utility of PCA in the context of environmental parameters, specifically related to water quality of the lakes.

Fig. 2
figure 2

Principal Component Analysis (PCA) of water from the selected lakes (i.e., Lake 1 = L1 (Oligo), Lake 2 = L2 (Meso) and Lake 3 = L3(Meso))

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In addition to PCA, Cluster analysis using Euclidean distance and non-weighted clustering using arithmetic mean (UPGMA) was conducted using PAST 3.0® software analyzing 14 physico-chemical parameters after the elimination of variables with commonality values lower than 0.6. The phenogram generated is shown in Fig. 3. The phenogram shows two distinct groups separating oligotrophic Lake 1 and mesotrophic lakes 2 and 3 as seen in Fig. 2.

Fig. 3
figure 3

Cluster Analysis of water quality parameters from the selected lakes (i.e., Lake 1 = L1 (Oligo), Lake 2 = L2 (Meso) and Lake 3 = L3 (Meso))

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Anatomical analysis of leaves of floating and submerged macrophytes using scanning electron microscope (SEM)

SEM analysis of adaxial surface of leaves of Eichhornia crassipes and Hydrilla verticillata from the selected lakes was conducted to analyze stomatal structure and distribution on the leaf structures collected. In mesotrophic lake (L3), submerged macrophytes Hydrilla verticillata were not found, due to overgrowth of free-floating macrophytes that affected the pelagic submerged floral diversity. Changes in macrophytic communities have been reported elsewhere due to anthropogenic factors, leading to overgrowth of exotic invasive macrophytes and extinction of submerged macrophytes (Poikane et al. 2018).

Adaxial surfaces of Eichhornia crassipes of Lake 1 (Samaspur Lake) show the normal anatomy with proper shaped and uniformly distributed stomata along with few deposits of biominerals on the surfaces and around the stomata, In contrast, the adaxial surfaces of the leaves of the Lake 2 (Kathauta) and Lake 3 (Haibatmau) showed an abundant deposition of biominerals on the leaves and around the stomata. In Lake 3 (Haibatmau) non-uniform distributions of stomata were reported on the adaxial surface of Eichhornia crassipes leaves.

The biomineral deposition on the adaxial surface and around the stomata of the leaves of Eichhornia crassipes seen in SEM images of the mesotrophic lakes L2 and L3 clearly indicates that they are transported from lake water through roots and that during the process of transpiration they are deposited around the stomata of leaves. High water hardness in Lake 2 and 3 (as shown in Table 2) correlated with the biomineral deposition. This shows that with increase in trophic state and deteriorating water quality, the anatomy of the leaf surface is affected with biominerals depositions. The shape, size and number of the stomata vary among the leaves of macrophytes present within the same trophic states as seen in Lake 2 and 3.

A variety of adaptations are developed in macrophytes tolerant to nutrient-enriched conditions, resulting in altered leaf characteristics to cope up with various environmental constraints. In tolerant macrophytes, there is often deposition of different inorganic salt on the leaf surface due to inorganic salt and nutrient excretion from glandular cells during the process of transpiration (Grašič et al. 2017). Macrophytes in high nutrient and salt conditions have developed a variety of adaptations with multiple functions that enable them to cope with environmental constraints (Flowers et al. 2015). In vascular aquatic plants, the main adaptive mechanism restricts the intake of excessive nutrients in the cell cytoplasm and in various other mechanism which relies on the leaf or stem succulence (Butnik et al. 2001) which affects the water management and cation concentrations, and manages nutrient redistribution within the different plant macrophytic organs (Flowers and Colmer 2008; Munns and Tester 2008). Excess nutrients are released through the leaf tissues into the atmosphere in volatile form (Larcher 2003). The strategy of macrophytes for salt and nutrient elimination depends on the concentration of nutrients in the lake water and is also based on the species-specific potential for this particular mechanism (Katschnig et al. 2013). Some of these adaptations in aquatic macrophytes such as epidermal structures, nutrient and salt excretion can alter leaf morphology and anatomy significantly (Grašič et al. 2017).

As a result of their constant submergence in polluted water, submerged macrophytes are highly affected by the water quality of the lake as compared to free-floating macrophytes, (Jamnická et al. 2006). In SEM analysis of stomata deficient, adaxial surfaces of Hydrilla verticillata leaves showed the deposition of biominerals. High deposition is observed in Hydrilla verticillata leaves of mesotrophic lakes (L2 and L3) (Supplementary Figs. 3 and 4) as compared to Hydrilla leaf surface of oligotrophic lake (L1) (Supplementary material Fig. 2). The water quality analysis showed maximum total suspended solids in the mesotrophic Lake 2 and 3 as compared to oligotrophic Lake 1 as shown in Table 2 which was further verified by PCA analysis (Fig. 1). Anatomical changes in leaves of Eichhornia crassipes and Hydrilla verticillata are shown in Figs. 2, 3 and 4 of supplementary material (Figs. 4, 5).

Fig. 4
figure 4

[SA] [SB] [SC] [SD] Transverse section of rhizome of the Eichhornia crassipes of the oligotrophic lake (Lake 1) showing the colorful crystals. [KA][KB][KC][KD] TS of rhizome of the Eichhornia crassipes from different sites of the mesotrophic lake (Lake 2) [HA] [HB]—TS of rhizome of the Eichhornia crassipes of the mesotrophic lake (Lake 3) as seen under the research microscope

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Fig. 5
figure 5

Scanning electron microscope (SEM) and Energy-dispersive X-ray spectroscopy (EDS) of rhizome of Eichhornia crassipes showing calcium oxalate and calcium carbonate crystals due to biomineralization A SEM of the transverse section of the rhizome of the Eichhornia crassipes of oligotrophic Lake 1 at 450–500 µm magnification B1 SEM images showing calcium oxalate crystal at 280–100 µm magnification B2 EDS spectrum of the B1 sample confirming calcium oxalate crystals C1 SEM images showing calcium oxalate crystal at 1,800–10 µm magnification C2 EDS spectrum of the sample C1 D1 SEM images showing shining calcium oxalate crystal in the rhizome at 500–50 µm magnification D2 EDS spectra of sample D1 E1 SEM images showing calcium oxalate at 20 µm magnification E2 EDS spectra of the E1 sample [Note- Arrow in the images shows the deposition of calcium carbonate crystals]

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Anatomical study of rhizome of floating macrophytes Eichhornia crassipes

Transverse Sections (TS) of the rhizome of Eichhornia crassipes of all the three selected lakes were analyzed under the research microscope and results are discussed below. The effects of nutrient enrichment on the rhizome anatomy of free-floating macrophytes are clear as they are responsible for the transport of minerals from the lake water to the upper macrophytic body of the plant.

Eichhornia crassipes from oligotrophic lake 1

TS of the Eichhornia crassipes of the oligotrophic Lake 1 show the presence of colorful crystals in the aerenchyma of the rhizome (Fig. 3[SA] [SB] [SC]). Some crystals were seen attached to the aerenchyma wall (Fig. 3[SD]). These depositions were unusual, as they were not reported in the two mesotrophic lakes. SEM and EDS analysis was carried out to determine the chemical composition and morphology of these crystals. The reasons for the variation in the size and color of these depositions (Fig. 3 [SA] [SB] [SC] [SD]) are not clear and have not been reported previously in the literature yet.

Eichhornia crassipes from mesotrophic lake 2

The invasive species Eichhornia crassipes was prominently present in the mesotrophic lake (L2). TS of the Eichhornia rhizome showed a normal anatomy under the research microscope (Fig. 3 [KA] [KB] [KC] [KD]), which indicates that they have developed tolerance mechanisms which resists anatomical changes (Bianchini et al. 2008). Tolerance in aquatic macrophytes is mainly determined from its transport mechanism across tonoplast and plasma membranes. The ecophysiological responses of aquatic plants help in their tolerance mechanisms (Tanwir et al. 2020). Defense mechanism activation against chelating substances and oxidative stress help in preventing biological damage (de Souza Reis et al. 2020). The ability to resist changes is associated with the development of intercellular strategies and different ecotypes dealing with the tolerance level in Eichhornia crassipes (de Souza et al. 2021).

Eichhornia crassipes from mesotrophic lake L3

TS of Eichhornia crassipes collected from mesotrophic Lake (L2) shows the normal anatomical structure shown in Fig. 3 [HA] [HB].

SEM and EDS analysis of rhizome of Eichhornia crassipes of oligotrophic lake 1 showing crystal formation

SEM analysis confirms the presence of crystals in the Eichhornia rhizome and EDS spectra confirm the presence of calcium oxalate and calcium carbonate crystals. Biomineralization phenomena are commonly observed in aquatic plants (Foster 1956; Franceschi and Horner 1980; Franceschi and Horner 1980; Franceschi and Nakata 2005; Borrelli et al. 2011). Calcium oxalate and calcium carbonate crystals are commonly found within idioblast near veins (Frank 1967; Cuéllar-Cruz et al. 2020). In Eichhornia crassipes calcium oxalate crystals are also found in the aerenchyma tissue (Kausch and Horner 1981; Xu et al. 2011; Cai et al. 2018; Cuéllar-Cruz et al. 2020).

In this study, calcium oxalate and calcium carbonate crystal formation in macrophytes was greater in the lesser-polluted oligotrophic lake (L1) compared to the two mesotrophic lakes (Lake L2 and L3). This shows that increasing trophic state and nutrient enrichment is not responsible for the formation of calcium oxalate crystals instead, crystal formation is due to high pH and alkalinity and lake morphometry is responsible for crystal formation within lake (L1) in the aquatic macrophytes (Downing 2010, Anderson et al. 2019, Cuevas et al. 2020). Water quality of oligotrophic lake (L1) shows high pH and low alkalinity. It seems that biomineralization and calcification in aquatic plants are brought about by shifting the inorganic carbon equilibrium in the lake ecosystem. The hardness of water within the lake L1 is the lowest among all the three lakes, indicating that biomineralization in aquatic plants is closely linked to photosynthetic inorganic carbon metabolism and less to calcium fluxes and calcium metabolism (Borowitzka 1984; Gwenzi 2019; Karabourniotis et al. 2020).

Role of biomineralization through aquatic macrophytes in carbon sink

Research in lacustrine ecosystem carbon dynamics and primary productivity often overlooks calcification in aquatic macrophytes that supports photosynthesis by making protons and forming carbon dioxide. Calcification causes higher decrease in a dissolved inorganic carbon (DIC) and prevents detrimental pH increase within aquatic ecosystem (Anderson et al. 2019).

$${\text{HCO}}_{3} ^{ - } \, + \,{\text{H}}_{2} {\text{O}}\, \to \,{\text{CH}}_{2} {\text{O}}\, + \,{\text{OH}}^{ - } \, + \,{\text{O}}_{2}$$
(1)
$${\text{Ca}}^{{2 + }} + \,{\text{2HCO}}_{3} ^{ - } \, + \,{\text{2H}}_{2} {\text{O}}^{ - } {\text{CaCO}}_{3} \downarrow \, + \,{\text{CH}}_{2} {\text{O}}\, + \,{\text{O}}_{2}$$
(2)

The availability of CO2 drops both by direct uptake of CO2 directly and by increasing the pH which accompanies photosynthesis. Increasing pH in the lake ecosystem changes the balance between all species of inorganic carbon which includes carbon dioxide (CO2), bicarbonate (HCO3 −) and carbonate (CO3 2−).Changes in form occur as CO2 is transformed to HCO3 − and eventually to CO3 2− as seen in Eqs. 1 and 2. This conversion challenges the ability of aquatic macrophytes to acquire CO2 for photosynthesis and uses carbon dioxide and bicarbonate required for their growth. Many aquatic macrophytes overcome this challenge by the process of calcification; that is by the formation of the CaCO3 crystals that accompany CO2 release, which help in meeting the inorganic carbon demands for photosynthesis in alkaline water (McConnaughey and Whelan 1997).

The coupling of photosynthesis and calcification seen in Eq. 2 is pH neutral. As a result photosynthesis can proceed without increase in pH (Sand-Jensen 1983, van den Berg et al. 2002., Sand-Jensen et al. 2018). The importance of calcification for sustaining photosynthesis has been documented in many freshwater macrophytes (Sand-Jensen et al. 2018), where it accompanies carbonate dissolution and carbon precipitation (Andersen et al. 2016). Calcification can occur spontaneously in the water column when regular photosynthesis (Eq. 1) drives pH upwards, converts bicarbonate (HCO3−) to Carbonate (CO32−) and induces formation of small calcite crystals when carbonate solubility is exceeded (Kalff 2002). It accompanies HCO3−utilization by aquatic macrophytes (Eq. 2) that precipitate carbonate in alkaline bands along cell surfaces by active calcium extrusion (McConnaughey 1991; Nõges et al. 2016). Shallow lakes which are rich in submerged macrophytes may accomplish these ideal conditions because they stratify into distinct layers on a daily basis. Stratification separates the upper photic epilimnion from the lower aphotic hypolimnion. The epilimnion is characterized by intensive photosynthesis, calcification and dissolve inorganic carbon, whereas the aphotic hypolimnion is characterized by intensive respiration and high CO2 concentration conducive to carbonate dissolution and dissolve inorganic carbon regeneration (Andersen et al. 2017). In a shallow water column, nocturnal surface cooling is sufficient to break the stratification by convection and allow DIC from bottom waters to mix with surface waters and support photosynthesis (Martinsen et al. 2019).

Calcification plays an important role in photosynthesis and carbon dynamics of aquatic ecosystems. Photosynthetic experiments with macrophytes confirmed that calcification rates were low at pH 7.5, while similar rates of calcification and carbon assimilation were attained when external pH approached 9.5 as carbon dioxide is saturated (Sand-Jensen et al. 2018, Anderson et al. 2019). Calcification plays the essential role in producing protons and making carbon dioxide available for assimilation from bicarbonate without further pH rise. This process is important because photosynthesis within macrophytes is markedly reduced as external pH approaches 9.5 (Christensen et al. 2013) and it usually stops entirely close to pH 10 (Sand-Jensen et al. 2018). Carbon is commonly the favored unit used in quantification of respiration and production as well as import, export and storage of organic matter in lake ecosystems (Wetzel 2001). Calcification can contribute to carbon dioxide oversaturation in lake waters and release to the atmosphere (Marcé et al. 2015). Biomineralization that causes formation of calcium oxalate and calcium carbonate crystals in plants represents a considerable carbon sink in the ecosystem with long residence time at both the ecosystem and global level (Anderson et al. 2019). This raises the possibility of exploring the role of calcium carbonate crystals in global carbon cycle and determining its effect on the climate change process (Tooulakau et al. 2016). Despite the role of carbon in biogeochemical cycling of carbon, calcium and silicon and their roles in sequestering atmospheric carbon. To date, less attention has been given in the literature to the impact of pollution and changing trophic state on biomineralization (He et al. 2014; Gwenzi 2019). Calcium carbonate and oxalate crystals play a major role in the physiological and structure functions of the macrophytes (Franceschi and Nakata 2005). Calcium oxalate and calcium carbonate crystals serve as a natural sink of calcium ions and carbon, thereby preventing calcium and carbonate accumulation in aerenchyma of the free-floating aquatic macrophyte Eichhornia crassipes (Prychid and Rudall 1999; Franceschi and Nakata 2005; Karabourniotis et al. 2020).

Calcium oxalate and calcium carbonate crystal formation in aquatic macrophytes has for some time been an important topic of research among plant scientists, even though the factors responsible for their formation and their functional role is still poorly understood (Nakata 2012; He et al. 2014). The main functions of calcium oxalate crystals reported in the literature are the regulation, balance and sequestering of calcium from the aquatic environment, ion balance, light collection and reflection, plant protection against herbivores and detoxification of heavy metals and oxalates (Libert and Franceschi 1987; Tooulakou et al. 2016).

The proposed pathways for oxalate production in aquatic macrophytes include photorespiratory glycolate/glyoxylate oxidation, hydrolysis of oxaloacetate and cleavage of ascorbate (Nakata 2003). Uneven distribution of calcium oxalate or calcium carbonate crystals has been reported in aquatic macrophytes (Jones and Ford 1972; Rahman et al. 2006). The current study confirms the presence of an unusual type of crystal idioblasts are found connected with aerenchyma. These crystals may protrude into the air spaces (Fran- ceschi and Horner 1980). Many studies consider that oxalate is the end product of the plant metabolism and that excess concentration may be toxic to the plants (Franceschi and Loewus 1995; Cuéllar-Cruz et al. 2020). Thus crystal and crystal forming idioblasts are a means of isolating this product in the macrophytic body and that is why they have been classified as the excretory idioblast in removing excess calcium (Foster 1956; Cai et al. 2018). Calcium oxalate crystallization may also help in removing excess oxalic acid, which is not toxic to plants, and may cause osmotic problems (Raven and Smith 1974). Aquatic macrophytes deposit their calcium carbonate crystal extracellularly as well as intracellularly in free-floating Eichhornia crassipes. As a result, the calcium oxalate concentration tends to increase as the plant matures (Torell et al. 2005). There is, however, evidences of calcium oxalate and calcium carbonate resorption in times of calcium depletion (Sunell and Healey 1979; Karabourniotis et al. 2020).

Energy-dispersive X-ray spectroscopy (EDS) of the submerged, floating and emergent macrophytes

Measurements of the roots of the macrophyte Eichhornia crassipes show the presence of more elements, due to their direct contact with lake water as compared to stem and leaves. Furthermore, leaves, stem and root of Eichhornia crassipes within oligotrophic lake 1 show fewer elements as compared to the macrophytes of mesotrophic lake 2 and lake 3. In submerged macrophyte Hydrilla verticillata leaves and stems within mesotrophic lakes (lake 2 and 3) and show greater number of elements in comparison to the macrophytes of the oligotrophic lake 1 (Fig. 5 of Supplementary material).

How does increasing pollution load and trophic state affect the elemental composition in the macrophytes?

The concentration of elements accumulated or present in macrophytic bodies is directly related to the concentration of that element present in the water and sediments of the lacustrine ecosystem (Guilizzoni 1991; Huang et al. 2017). Quantitative assessment of lacustrine pollution shows the relevant correlation between concentration of the elements in the lake water to the concentration of the elements present in the aquatic macrophytes (Guilizzoni 1991; Krems et al. 2013). Ion exchange is the main process by which absorption of metals takes place via surface of aquatic macrophytes (Schneider et al. 2001). Change in water chemistry due to increasing pollution load and trophic state, will affect the elemental composition in the free-floating, submerged and emergent macrophytes in the lake ecosystem. Quantitative assessment of lacustrine pollution indicates a correlation between the concentration of the elements into the lake water and the concentration of the elements present in the aquatic macrophytes (Krems et al. 2013).

Elemental bioabsorption processes in the macrophytic cells occur through surface absorption as well as through intracellular and extracellular accumulation (Veglio and Beolchini 1997, Moe et al. 2019, Dalla Vecchia et al. 2020). Bioabsorption of elements in macrophytes is defined as a reversible and fast process that is based on physico-chemical binding of metal cations through ion exchange, adsorption, microprecipitation, complexing and chelation.

Elemental variation in free-floating, submerged and emergent macrophytes varies with the trophic state of the lakes. Floating and submerged macrophytes contain higher elemental concentrations of cations than the emergent macrophytes. Increases in trophic state and pollution lead to accumulation of cations with macrophytes.

Conclusion

This study shows that within the Central Gangetic Plain, trophic State Index provides a comprehensive tool for water quality assessment of freshwater lakes. Furthermore, the study has shown how the increasing trophic state and deteriorating water quality affect the anatomy of macrophyte leaf surfaces through biomineralization of their surfaces. Anatomical changes in macrophytes shown in this study include effects on the leaf surface, changes in stomatal structure and depositions around it. It is clear from the study that within mesotrophic lakes such as L2 and L3, nutrient enrichment and pollution stresses affect the leaves of the macrophytes through the accumulation of biogenic materials, whereas in oligotrophic lake L1 macrophytes show normal morphology with proper stomatal structures.

The anatomies of rhizomes of free-floating Eichhornia crassipes in lakes L2 and L3 were found to be normal in spite of being in mesotrophic condition and polluted states of the lake. In contrast within the oligotrophic L1, the presence of calcium oxalate and carbonate crystals was noted within macrophytes. Environmental and genetic factors are the main factors responsible for biomineralization within aquatic macrophytes (Bauer et al. 2011). Water quality of oligotrophic lake (L1) shows high pH and low alkalinity. Biomineralization and calcification in Eichhornia, therefore, appear to be mainly brought about by achieving supersaturation of the lacustrine ecosystem, which increases carbonate ion activities by shifting the inorganic carbonate ion equilibrium. The main functions of calcium oxalate and calcium carbonate crystals in aquatic ecosystems are regulating cytoplasmic free calcium levels in the macrophytes. They also help in biogeochemical cycling of carbon, calcium, and silicon and in the sequestration of atmospheric CO2, alleviating both salt and temperature stresses. It has also been reported that they regulate ion balance (e.g., sodium and potassium), detoxifying oxalic acid, and reduce alkalinity generated by nitrate assimilation (He et al. 2014).

The study has also shown that the trace elemental concentration within aquatic macrophytes varies with change in trophic state from samples collected from different lakes. Macrophytes of the mesotrophic lakes contain higher trace elemental activities compared with the macrophytes within oligotrophic lake. The study shows that the trace elemental concentrations in floating macrophytes are higher than in submerged and emergent macrophytes. It seems, therefore, that the trophic state and water quality are the principal determinants of the elemental variation in aquatic macrophytes.