Evaluating Potential of Traditional and Newer Periphytic Diatom Metrices for Deciphering The Impact of Organic Pollution on Residing Aquatic Biolms

The present study examined impacts of organic contamination on periphytic biolms at different sites near rivers using various traditional and newer diatom-based metrics, which is rarely tested under organic contamination. All examined sites were dominated by diatoms rather than green algae, which often dominate nutrient-rich sites. Biodiversity indices were higher at riverine and mass bathing sites than the kitchen waste site. Newer diatom metrics showed more promising results in terms identifying possible differences among sites. The three examined waste sites showed the dominance of motile forms, high percentages of unhealthy cells and enlarged lipid bodies in the community, indicating the contamination of nutrients and organic matter. A remarkable higher percentage of tube-dwelling forms at kitchen waste sites likely indicates the presence of organic contamination in the water. The percentage of deformed diatom frustules was low at all sites, indicating that organic contamination did not affect the morphology of diatom cells, but the occurrence of both cell shape and striae deformations at the RWS site might indicate slight metal contamination. Finally, we conclude that the combination of both traditional and newer diatom metrics in biomonitoring improves our understanding on the effects organic contamination in aquatic ecosystems.


Introduction
In India, the population is increasing day by day, which affects our waterbodies at an increasing rate (Paul, 2017;Dwivedi et al., 2018). Increasing population leads to rapid urbanization and large-scale industrialization in the riverine regions of uvial ecosystems. Due to a combination of public negligence and oblivious governmental agencies, water pollution is a serious problem in India (Rai et al., 2013). All major rivers in the Ganga Plain in India face issues with pollution (Paul, 2017). Water pollution leads to various problems, including algal bloom formation, health hazards, aversion by tourists and death or impairment of aquatic fauna (Rai et al., 2013;Dwivedi et al., 2020).
Water quality assessment of waterbodies is usually done by sophisticated instruments that measure water chemistry, such as atomic absorption spectroscopy (AAS) and inductively coupled plasma (ICP). Generally, these instruments are costly, need highly skilled operators and involve tedious sample In the light of the problems with using only chemical testing, biomonitoring practices are becoming increasingly used (Pandey et al., 2014). Biomonitoring relies on the status of residing biota in waterbodies and on this basis, provides much more effective information about the overall ecological status of waterbodies (Pandey et al., 2018a,b). In comparison to traditional chemical methods, biomonitoring shows more promise, as it is relatively easy, cheap and provides cause-effect relationships Various organisms (bacteria, shes, micro and macroinvertebrates, aquatic plants etc.) are regularly used for aquatic biomonitoring (Bettinetti et al., 2012). Among these organisms, algae are perhaps the most used on a global basis. Reasons contributing to the widespread use of algae are their often cosmopolitan distribution (especially at the genus level) and autotrophic nature, short-life spans, easy availability and the generally low expense to collect and process samples. Among algae, diatoms are especially used for assessing the water quality worldwide. In addition to the advantages of using algae for biomonitoring, diatoms have several characteristics that make this group particularly useful, including their robust silica frustules, high biodiversity with a range of environmental tolerances and short life-span. This short lifespan makes them highly sensitive to many types of environmental and anthropogenic stresses, and This study examined the ecological health of various types of polluted sites in the Moradabad and Bareilly districts of Uttar Pradesh, India, using a combination of traditional (taxonomic) and newer diatom (non-taxonomic) metrics.

Sampling sites
Various organically polluted sites (mass bathing, kitchen wastes and riverine sites) near MJP Rohilkhand University and several sites from Moradabad were examined and periphytic diatom samples were collected. MJP Rohilkhand University men's and women's hostels were chosen for collecting mass bathing and kitchen waste area samples. Sampled sites on the Ram Ganga River in Moradabad (Figs. 1 and 2) include sites with a combination of point-source and more diffuse pollutant sources (Table 1). All the sites were selected on the basis of their long term legacy of organic pollution.

Statistical analysis
Data (Shannon index, species richness, % deformities and total deformities) were analysed using oneway analysis of variance (ANOVA) followed by Tukey's HSD test, as appropriate. "PAST" software (Natural History Museum, University of Oslo) was used for data analysis and for calculating the Shannon index for algal samples from different sites.

Shannon index and species richness
A total of 45 algal species were identi ed (34 diatom, 9 green algae and 2 euglena species) at the riverine waste sites ( Figure 5).

Discussion
In the present study, three speci c, nutrient-enriched waste sites were characterized using a variety of traditional (taxonomic) and newer diatom (non-taxonomic) metrics to assess the potential of these metrics for biomonitoring. Three classes of algae dominated the investigated waste sites: Bacillariophyceae; Chlorophyceae and Euglenophyceae. Of these three classes, Bacillariophyceae were the most abundant at all three waste sites. This nding was not in agreement with the ndings of Palmer (1969), who reviewed more than 200 research papers dealing with organic contamination and algal diversity. Palmer (1969) reported numerical dominance of Chlorophyceae, followed by Bacillariophyceae under conditions of organic pollution. The difference in dominant algal class with organic pollution is likely due to the type of algal community sampled in our study, in which we sampled periphytic algal communities. These substrate-associated communities are considered permanent residents of the waterbodies due to their attach nature. In contrast, many green algae are phytoplanktonic and most attached forms are lamentous. In contrast, periphytic diatoms have the excellent adhesive properties and can attach to almost any available substrate and thereby dislodgement due to normal currents found in the uvial waterbodies. In terms of genera, Palmer (1969) reported the dominance of Euglena (Euglenophyceae), and Chlamydomonas, Scenedesums and Chlorella (Chlorophyceae) at organically polluted sites. In contrast, we found that the diatom genera Nitzschia and Navicula dominated at all three waste sites., whereas Palmer (1969) ranked these genera in the 6th and 7th places, respectively. At the species level, the riverine waste sites (RWS) had high abundance of Euglena gracilis and Nitzschia palea, species that Palmer (1969) considered characteristic of contaminated sites. Findings at MBS and KWS sites similarly had high abundance of Nitzschia palea but Euglena gracilis was low in comparison to diatom species. In our view, we think that ndings of Palmer (1969) need further examination, especially incorporating information about the type of algal community (phytoplanktonic, periphytic and benthic) sampled and how these habitat-based communities might produce different results. Better understanding of habitat differences will contribute toward developing more effective biomonitoring strategies for organic pollution. Riverine waste sites (RWS) had signi cantly more deformities in diatom frustules at than MBS and KWS, although the overall rate of deformities was not high (less than 0.5%). The main reason for this higher percentage of deformed diatom frustules at RWS is that riverine waste sites bear the legacy of cocktail of pollutants (nutrients, organic matters, metals etc.) coming from household discharges, agricultural runoff, discharges from waste treatment plants and metals discharge from brass factories. Thus, the cumulative effect of these toxicants results in a higher percent deformed frustules at riverine waste sites. Generally, high rates of deformed frustules are reported under metal stress. For example, in a laboratory mesocosm experiment, Cu and Zn exposure produced 6% and 8% deformed diatom frustules (Pandey et al., 2015). Our results indicated that the three investigated sites are all affected by organic matter enrichment, but may also be affected by other contaminants (e.g., nutrients and metals). Presence of elevated nutrients results higher cell growth (cell division), which may enhance uptake of other contaminants (metals, pesticides, insecticides), which is manifested in the dominance of nitzschoid diatoms, higher unhealthy cells, induction of lipid bodies (higher number, size and biovolume) and the presence of deformed cells.

Conclusions
Traditional biodiversity methods indicated some impairment, with the three sites ordered from best to worst as: MBS > RWS > KWS, based on species richness and the Shannon index. The non-traditional diatom metrics presented a more complex picture of the three sites -enlarged lipid bodies in some diatoms and dominance by motile diatoms at all sites indicated stress and nutrient/organic matter contamination, respectively -but other metrics differentiated the sites. RWS had a higher rate and two different types of deformities in diatom frustules, indicating possible metal contamination. The high rate of tube-dwelling diatoms at KWS may result from high nutrient loads or speci c contaminants (e.g., herbicides or fungicides). MBS (the 'best' site as indicated with traditional metrics) also had the highest proportion of unhealthy diatoms (based on chloroplast and cytoplasm condition), which may indicate slower diatom turnover because of relatively lower nutrient concentrations or uctuating nutrient availability compared to the other two sites.
Incorporating newer, non-traditional diatom metrics provided additional evidence about the types of stresses experienced by the biota at each site. Because these diatom metrics are also relatively faster, less expensive, and require less expertise, developing biomonitoring strategies that incorporate both traditional and non-traditional metrics is cost effective and will increase understanding of both the ecological heath and possible reasons for impairment.

Declarations Declaration of con ict of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in in this paper.
CRediT authorship contribution statement