TAXONOMIC INVESTIGATION OF EUPLANKTONIC DIATOM COMMUNITIES AS INDICATOR OF COPPER IN THE BANK OF THE SUBARNAREKHA RIVER, GHATSHILA, JHARKHAND, INDIA

The aim of this study was to demonstrate and evaluate the diatom communities in the copper infected areas readily associated with the Hindustan Copper Limited (HCL) at the bank of the Subarnarekha River. This study was based on three sampling sites commonly designated as high copper (>100 μg.L-1), medium copper (≤100 μg.L-1) and low copper (≤50 μg.L-1) contaminated area. Results indicated the detailed taxonomic description of 31 species that are dominant or less dominant over these contaminated area. Among the identified taxa, 10 were recorded as new to the Jharkhand state. Water analysis has suggested the presence of 17 species in the high copper contaminated area adjacent to HCL. Nine species was less dominant in the outlet of HCL that belonged to the medium contaminated and only 5 species were dominant over the low copper contaminated area. Physico-chemical parameters like pH, air and water temperature, salinity, conductivity, light extinction coefficient, turbidity, dissolved inorganic salts, dissolved oxygen and carbon-di-oxide, biological oxygen demand and total hardness were also estimated in the copper contaminated sites. Relatively all the species of Cymbella and Navicula were associated with high copper accumulation. Most interestingly, one harmful species Halamphora coffeiformis, which was recorded as most dominant species in high copper exposed area, has shown to be the best copper tolerant and copper indicator species.


INTRODUCTION
Biosorption is a well-known method of phytoremediation, which binds the toxic chemicals or metals and accumulates it in their biological systems especially in cellular structures. The process is widely used to encourage the remediation of heavy metals from the aquatic ecosystems. It also has the potential towards wastewater treatment. Metal toxicity in aquatic ecosystems is commonly triggered by anthropogenic activities including domestic and industrial wastewater, agricultural runoff and dumping of toxic chemicals, e-waste and others (Satpati, 2021). The deposition of toxic elements or trace metals in the water bodies resulting in severe environmental impacts including contamination of surface and ground water and increasing the rate of biomagnification (Sbihi et al., 2014;Satpati, 2021). Trace metals like copper (Cu) is a well known aquatic pollutant for its adverse affects on phytoplanktons, especially diatoms (Absil, Kroon & Wolterbeek, 1994). The heavy presence of Cu in the aquatic food chains may be hazardous to the associated living organisms and to the environment (Nor, 1987). Aquatic living systems may scavenge the trace metals from the water column as well as from the bottom sediments or from both. Recently, algae have served as the most potential aquatic living system or bioindicator for accumulating toxic metals (Zeraatkar et al., 2016). Diatoms belong to the group of Bacillariophyta (Guiry in Guiry & Guiry, 2021, AlgaeBase), which are frequently used as bioindicators for heavy metals in aquatic bodies. They are unicellular having silicified cell wall. The cell wall consists of two valves held together by a band of girdle. Most of the studies have been done so far on taxonomic documentation. In India, there are many reports on the freshwater diatom flora with detailed taxonomic account (Gandhi, 1959(Gandhi, , 1967 (2014) have studied the morphological changes of few diatoms exposed to Cu, lead (Pb) and zinc (Zn). Modification of raphe was found more frequent in Fragilaria capucina, Gomphonema parvulum, Nitzschia palea, Pinnularia conica and Ulnaria ulna. As diatoms are planktonic, they remain in the open water systems rather in the sediment (Cattaneo et al., 2011). Diatoms are ecologically diverse from centric to pennate form and found in almost all microhabitats in the aquatic ecosystems (Arguelles, 2019). Diatom assemblages can be formed in the open water systems of rivers, lakes and canals (euplanktons), they may be associated with plants (epiphytic), they may be found in the sand (epipsammon), or mud (epipelon) and even in animals (epizooic) (Dixit et al., 1992;Arguelles, 2020). In the present research, the work has been carried out on the taxonomic investigation of some euplanktonic diatoms, which frequently dominate over the Cu mining area. Hindustan Copper Limited (HCL), situated at the bank of the Subarnarekha River of Ghatshila, is the biggest source of Cu discharge in the surrounding aquatic habitats. Cu mining wastes flow directly into the river without any treatment, resulting in significant growth of diatoms and other planktonic organisms. The sampling sites were chosen on the basis of high, medium and low Cu contamination. Diatom assemblages of these sites were identified and described in detail in relation to abundance. The dominant species from the three different sampling stations were marked on the basis of abundance. Physico-chemical parameters like nitrate, phosphate, silicate, sulphate, calcium, dissolved oxygen (DO), biological oxygen demand (BOD), conductivity, salinity and pH were also recorded in the present study. The objective of this study was to determine the diversity of diatom flora as indicator of Cu in the adjoining water bodies of HCL and Subarnarekha River. In addition, the abundance of the diatom species in terms of low, medium and high Cu accumulation were also examined. The detailed taxonomic description suggests the proper identification of the euplanktonic diatoms as pollution indicator in aquatic ecosystems. The biochemical assessment of the water has also determined the water quality in the adjacent water bodies of HCL and Ghatshila.

Sampling sites
For the collection of diatom and water samples, four sites were chosen: canal adjacent to HCL, outlets of HCL poured into the Subarnarekha River and the The aim of this study was to demonstrate and evaluate the diatom communities in the copper infected areas readily associated with the Hindustan Copper Limited (HCL) at the bank of the Subarnarekha River. This study was based on three sampling sites commonly designated as high copper (>100 μg.L -1 ), medium copper (≤100 μg.L -1 ) and low copper (≤50 μg.L -1 ) contaminated area. Results indicated the detailed taxonomic description of 31 species that are dominant or less dominant over these contaminated area. Among the identified taxa, 10 were recorded as new to the Jharkhand state. Water analysis has suggested the presence of 17 species in the high copper contaminated area adjacent to HCL. Nine species was less dominant in the outlet of HCL that belonged to the medium contaminated and only 5 species were dominant over the low copper contaminated area. Physico-chemical parameters like pH, air and water temperature, salinity, conductivity, light extinction coefficient, turbidity, dissolved inorganic salts, dissolved oxygen and carbondi-oxide, biological oxygen demand and total hardness were also estimated in the copper contaminated sites. Relatively all the species of Cymbella and Navicula were associated with high copper accumulation. Most interestingly, one harmful species Halamphora coffeiformis, which was recorded as most dominant species in high copper exposed area, has shown to be the best copper tolerant and copper indicator species. river itself commonly designated as Station 1 (22.5954° N, 86.4519° E), Station 2 (22.5962° N, 86.4522° E) and Station 3 (21.3325° N, 87.2341° E) respectively. All sampling stations are situated in Ghatshila, Jharkhand (Figure 1).

Diatom collection and preservation
The copper containing sites associated to Subarnarekha River was investigated in March 2018. The diatom sample was collected through the phytoplankton net of mesh size 25 μm . After collection, the turbid sample was brought to the laboratory and centrifuged at 10000 rpm for 10 minutes. The pellet thus collected was preserved in 4% (v/v) formalin for the microscopic study. All the preserved materials were assigned to Calcutta University Herbarium (CUH) voucher specimens.

Water analyses
Water samples were collected in triplicates at the depth of 0.5 m. The physicochemical parameters were determined with the help of the filtrate obtained from the water samples. All parameters like pH, temperature, electrical conductivity, total hardness, light extinction coefficient, BOD, salinity, nitrate, phosphate, silicate, sulphate and calcium were analyzed using the standard methods of APHA (APHA, 1998). Salinity, pH and temperature were recorded immediately after sampling with ERMA Refractometer (ERMA, Tokyo), Beckman potentiometer zeromatic II and centigrade thermometer respectively. DO content in water sample was estimated in situ following Winkler's Iodometric titration method (Winkler, 1888).

Light Microscopy and Identification
For light microscopy study, slides were prepared with 20% glycerin (v/v) and photographs were taken under Carl Zeiss Axioster Plus Microscope by Cannon Power Shot 500D Camera with a coupled micrometer eyepiece (Satpati et

Cu accumulation study
Cu accumulated in water samples were analyzed using an ICP 2070 Spetrophotometer (Baired, USA) and AAS using a Varian Spectr AA10 apparatus with Graphite Tube Atomizer GTA-95 (Victoria, Australia). The measurement accuracy was checked by the reference of Chmielewska & Medved (2001).

Water analyses
Water analysis report is demonstrated in table 1. During the study, all the sampling stations showed a static air temperature but slightly varied in water temperature. Water temperature was recorded minimum in station 1 with 28.35°C whereas highest in Subarnarekha River (station 3) with 30.33°C. pH ranges from slightly acidic (below 7.0) to slightly alkaline (above 7.0). The water pH of the canal adjacent to HCL (station 1) was recorded 6.8. However pH was recorded highest (7.3) in Subarnarekha River. High turbid condition of the water was noticed in station 1 followed by station 2 and 3. Electrical conductivity and light extinction coefficient was significantly decreased in the order station 3>station 2> station 1. Highest conductivity recorded in Subarnarekha River was 560.33 μ S.cm -1 . Total hardness varied from 260.33 in station 1 to 180.35 in station 3. Salinity was recorded highest (10 ppt) in station 1 and lowest (6 ppt) in station 3. Comparatively DO was highest in station 3 with 3.42 mg.L -1 and lowest in station 1 with 2.21 mg.L -1 . However, station 1 recorded highest amount of dissolved CO2 and BOD instead of station 2 and 3. Interestingly nitrate, sulphate and silicate level in the water was high in station 1, which was highly polluted and found adjacent to HCL. Phosphate level in the water of Subarnarekha River was recorded highest (0.84 mg.L -1 ) and lowest (0.57 mg.L -1 ) in the outlet of HCL poured directly into the river. Relatively the concentration of the calcium was high in station 3 and low in station 1. Accumulation of Cu in the water body was recorded highest in station 1 (400 μg.L -1 ) and lowest in station 3 (47.87 μg.L -1 ).

Diatom composition
In the present study, a total number of 31 species were investigated, which belong to 16 families, 10 orders under the class Bacillariophyceae. The diatom composition has suggested the dominance of Cymbella with 6 species followed by 3 species each of Nitzschia and Rhopalodia in the Cu contaminated area. Two species each from the genus Navicula, Pinnularia, Amphora and Synedra were also documented from the study sites. A large number of species were documented as Cu indicator or tolerant in Station 1, closely associated canal of HCL. From Table 2 it can be obtained that, 17 species are rich in Cu in Station 1 of which Halamphora coffeiformis was found to be most dominant over the area. Similarly this species was absent in station 2 and 3. Interestingly all species of Cymbella and Navicula were reported as high Cu tolerant species (Table 2). In station 2, nine diatom species were dominant of which both the species of Pinnularia, P. acrosphaeria and P. viridis showed positive response to Cu accumulation. Two species each of Rhopalodia and Nitzschia were recommended as Cu tolerant species in Station 2. However in station 3 only 5 species dominated as Cu tolerant upto 50 μg.L -1 . Both the species of Synedra, S. ulna and S. ulna var. amphirhynchus were designated as Cu indicator species in Subarnarekha River. Among the identified diatom species, 10 species viz., Halamphora coffeiformis, Rhopalodia gibberula, Mastogloia smithii var. lacustris, Nitzschia nana, Himantidium minus, Synedra ulna var. amphirhynchus, Fragilaria intermedia var. robusta, Grammatophora undulata, Diatoma mesodon and Ctenophora pulchella were recorded as new to the Jharkhand State.  Valves semi-lanceolate, dorsal margins, ventral linear, slightly concave with rostrate or capitate apices, 4-6 times longer than broad, 30-50 μm long and 4-12 μm broad; raphe straight, excentric; striae dorsal, coarse, radiate, 8-12 in 10 μm area.
Limnological parameter greatly influences the assemblage of diatoms and reduces water quality (Bigler & Hall, 2002). It has been suggested that the diatoms respond well to hardness, alkalinity, pH, salinity and nutrients (Greenaway et al., 2012). The appearance of low pH in water bodies indicates acidification. In our study we have observed the pH value in station 1 was 6.8, which is slightly acidic suggesting the starting point of acidification due to pollution (Petrou et al., 2019). High acidification also diminishes silica production in diatoms (Petrou et al., 2019). Minimum value of calcium in station 1 suggests the low pH. Total hardness and turbidity values in all stations suggest mineralization and eutrophic condition of the water bodies. Direct pouring of mining wastewater into the canals, outlets and in the River of Ghatshila results in significant changes to nitrate, phosphate, sulphate and silicate levels. High nitrate and sulphate values indicate the eutrophic condition of the water bodies of the sampling sites either due to anthropogenic activities or industrial pollution (Bella et al., 2007). Diatom communities play a significant role in maintaining the water quality in India. Some workers have concluded that diatoms can serve as indicator of organic and anthropogenic pollution (Choudhury & Pal, 2010). The photosynthetic activity of the diatoms is directly associated with DO of the water. The productivity in relation to gross primary productivity (GPP) and net primary productivity (NPP) is directly correlated with DO. Rise in DO level in water results in high GPP, whereas rise in BOD is a measure of drop in NPP in the concerned water bodies. In our study DO value sharply increased in the order: station 1 > station 2> station 3, indicating the pollution level in the water. Similarly BOD level decreased in the order: station 1 < station 2< station 3. As diatoms are primary producer they respond quickly to the ecological perturbations such as changing n physico-chemical parameters due to pollution (Adon, Quattara & Gourene, 2012). High value of dissolved CO2 in station 1 adjacent to HCL indicates the competition of zooplanktons and other aquatic animals with the diatom communities for respiration. Poor water quality of station 1 due to contamination of Cu suggests diatom shift to the neighboring area of station 2 and 3. At some places diatoms can tolerate high metals whereas it drastically changes species composition in other places (Cattaneo et al., 2011).
The conversion of oligotrophic to mesotrophic environment results in changing of species composition and species richness (Cumming et al., 1995). In our study it was sharply observed that the dominant species in station 3 vanished completely in station 1, which was highly polluted with Cu (Table 2). Similarly, species of highly Cu infected area were not observed in the less polluted area (station 3). Hence our study mainly focused on taxonomic implications of diatoms in Cu infected area with changing water quality.

CONCLUSION
Study of euplanktonic diatom communities in Cu mining sites is scarce. The present study was undertaken as first documentation of diatom communities in Jharkhand, India. The site is rich in Cu as it is situated adjacent to HCL on the bank of Subarnarekha River. A total number of 31 diatom species were documented from three sampling stations distinguished by high, medium and low Cu contamination. Maximum species dominated in station 1, which is closely associated to HCL. The taxonomic shifts were sharply noticed in three sampling sites. Oligotrophic to mesotrophic habitat suggests the eutrophication, acidification and mineralization of the water ecosystems. Physico-chemical parameters show the condition of the water bodies due to Cu contamination. From the study it can be concluded that the diatom assemblages of Ghatshila can serve as best bioindicator of Cu.