In the present study, three specific, 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 finding was not in agreement with the findings 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 filamentous. 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 fluvial 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 findings 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.
Biodiversity metrics are regularly used for biomonitoring purposes (Pandey et al., 2017). Species richness and Shannon index values of the sampled algal communities had significantly lower values at KWS versus the significantly higher values found at MBS and RWS. Lower index values at KWS may be due to particular contaminants associated with kitchens, such as insecticide and pesticide contaminants (from washing of vegetables and fruits) or soaps and detergents that accompany the nutrient enrichment. Shannon index value >3 is considered as clean water quality while values between 1-3 are considered as moderately polluted and value <1 is considered polluted (Gökçe, 2016). On this basis, MBS sites (3.2) are relatively clean while RWS (2.8) and KWS (2.1) sites are moderately polluted. However, field observation of these sites is at odds with these results. RWS, MBS and KWS have long history of organic contamination, as they are continuously polluted by their respective discharges (soaps, detergents, toothpastes, shampoo discharges, cooking oils, waste foods, discharges from ritual practices etc.) since from their formation as drainages. Soininen et al. (2012) also reported species richness as a single metric may provide incomplete information about aquatic ecosystems. In the same context, Blanco, (2012) examined 640 sites of France and found poor linear correlations among environmental factors indicating ecological status and came to the conclusion that biodiversity indices provide inconsistent information about the known impairment of the waterbodies. Biodiversity indices-based analysis is strongly dependent upon cell counts and the correct taxonomic identification of algae. The indices are susceptible to change with any environmental and anthropogenic disturbance.
Diatom life-forms (benthic, planktonic, motile, colonial, tube-dwelling, stalked and adnate) can have application in biomonitoring. Different life-forms of diatoms provide different strategies to resist or tolerate some environmental and anthropogenic stresses. In the present study, all sites were dominated by motile forms. Motile forms may be associated with: (1) the secretion extracellular enzymes that allow motile diatoms to more easily use macromolecules adsorbed on substrates (Pringle, 1990). (2) the larger size of motile diatoms relative to adnate diatoms make them better equipped to store more nutrients. (3) motile diatom forms can easily move from nutrient deficient microenvironments to nutrient enriched locations (Johnson et al., 1997). KWS sites also had a high abundance of tube-dwelling diatom forms, which were only common at this site. Tube dwelling diatoms have been reported in nutrient and organic-matter contaminated sites (Marcel et al., 2013). Fricke et al. (2017) reported higher dominance of tube-dwelling diatom (Berkeleya rutilans) as an indicator of eutrophication in sedimentary marine habitats of Argentinean waters. Tube dwelling diatom forms have also been reported under herbicide and fungicide exposure in a lotic mesocosm experiment (Rimet and Bouchez, 2011). In the present study, kitchen waste sites can be contaminated with pesticides and herbicides, which comes from the washing fruits and vegetables. RWS sites are more uniform in distribution of different life-forms than KWS and MBS sites. Understanding the role of various life-forms within biofilms enables better understanding of species-environment relationships and interactions, which is critical for developing effective biomonitoring strategies for aquatic ecosystem (Rimet and Bouchez, 2011; Pandey et al., 2017).
Observations of live samples of periphytic diatoms provide valuable information about the ecological status of the collection sites (Pandey et al., 2017). Fresh samples include healthy (live), unhealthy and dead diatom frustules in different proportions. Diatom cells with intact, distorted and empty protoplasmic content are considered as healthy, unhealthy and dead diatoms, respectively (Agusti et al., 2015; Pandey et al., 2018). In the past several years, several authors have used diatom health as an indicator of ecological health of waterbodies (Pandey et al., 2017, 2018; Werdel et al., 2021). In the present study, KWS and RWS sites showed higher dominance (> 50%) of live (healthy) frustules than unhealthy and dead frustules, possibly because of the availability of nutrients for the diatom cells at these sites. Similarly, a higher proportion of healthy diatom cells was also examined in eutrophic lakes of Florida, USA. High nutrients may support diatom cell reproduce at a faster rate, which produces new and rejuvenated cells regularly. These sites also bear contamination of pesticides, herbicides and metals (at RWS), which can result in unhealthy cells even in the presence of nutrient enrichment. Wood et al. (2014, 2016) reported visible changes in diatom chloroplasts under exposure to different types of herbicides (atrazine, simazine, hexazinone, tebuthiuron, diuron, MCPA, 2,4-D and glyphosphate). Debenest et al. (2008) also reported alteration in chloroplast morphology under herbicide exposure. Alteration in chloroplast morphology can also result from metal stress (Licursi and Gómez, 2013: Pandey and Bergey, 2016). At MBS sites, healthy diatom cells had lower abundance than unhealthy diatom cells. This may be due to intermittent availability of nutrients, as nutrients are regularly flushed out, often creating nutrient deficient conditions. Nutrient depletion was also associated with deteriorated cell status in bloom forming diatoms, which is associated with large sinking events in the Arctic (Svalbard Islands) region (Agusti et al., 2018).
Different algal groups are classified partly on the basis of the type of reserve food materials. In diatoms, the chief reserve food is in the form of lipid bodies (LBs). Under stress, LBs in diatoms may show increase in number and percent biovolume contribution per cell. In the present study, LBs number and its biovolume contribution per cell showed appreciable changes, especially in Nitzschia and Navicula species. Higher abundance of Nitzschia species is regularly reported under organic contamination but the information regarding LBs is still lacking. However, the present study showed that contamination of nutrients and organic matter may also affect LBs. Among the investigated diatom species, Nitzschia palea and Achnanthes exigua had normal lipid body numbers but their biovolume contribution per cell was appreciably elevated, i.e., up to16% and 30%, respectively. Induction of LBs in various diatom species is well documented under metal stress. Higher abundance of LBs in Achnanthidium exiguum (Achnanthes exigua) was reported under metal stress, as was increased LB percent biovolume. i.e., 20-77% and 13-53% under Cu and Zn stress, respectively. Pandey et al. (2018) reported higher abundance of LBs in Nitzschia palea under metal (Cu, Zn and Ni) stress, i.e., 8-25%. Similarly, Pandey and Bergey (2018) reported 50-60% LBs contribution per cell under Cu and Zn stress in the River Ganga, India. Extra LBs (in number and biovolume) may help diatoms combat stress and provide an additional food source. LBs in diatoms also affect buoyancy (Smetacek, 2001) under stress conditions and may enable them to maintain a proper depth in the water column, as sinking is associated with cell death (Agusti et al., 2018). Also, by helping regulate buoyancy, LBs inside diatom cells assist in cell movement (Wang et al., 2013), which may help diatoms move from nutrient deficient microenvironments to nutrient enriched conditions. Excess nutrients in the aquatic environment can support bloom formation of diatom cells, and as a result, a rapid decrease in nutrient concentration in the water column takes place, which may result in the poor health of live diatoms (shrinkage of protoplasmic content) and an associated enhancement of LBs in response to nutrient stress. LB induction in diatom samples from all three riverine waste sites clearly indicate stress, presumably associated with the high (and possibly changing) nutrient loads.
Diatoms are well known for their silicon cell wall, which is faithfully reproduced through the generations. Silicon frustules also bears species-specific ornamentation (especially striae and the raphe), which are extremely useful in the identification of diatom species. Any change in the normal morphology (outline, striations and raphe) of diatom frustules considered deformed. In the present study, deformed frustules were found in Navicula saprophila, N. subminuscula, N. slesvicensis, Nitzschia palea, N. minuta, Gomphonema parvulum and Melosira distans. Deformities in diatom frustules are only occasionally reported under nutrient and organic stress. Dziengo-Czaja et al. (2008) similarly reported deformities in diatoms under organic matter load (nitrate and phosphate) in the Western Puck Bay of the Baltic Sea, but the reported species differed from those we found because Dziengo-Czaja et al. (2008) worked in a marine rather than our freshwater environment and the two environments have little species overlap (Pandey et al., 2018). In comparison to fresh water, marine waters showed relatively bigger size of diatom species (Litchman et al., 2009) due to which the chances of getting deformed frustules under such conditions get increased.
Riverine waste sites (RWS) had significantly 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). Field conditions had comparable deformity rates, with 8.16% (Zn) and 14% (Cu) deformed diatom frustules from severely metal polluted metalliferous sites of Zawar and Khetri (Pandey et al., 2016) and 6-8% deformed frustules under metal contamination (Cu, Zn and Ni) at the Soyo site in the Han River, South Korea. In the present study, deformities were more common in raphid diatoms than araphid diatoms, although araphid diatoms generally show higher percentages of deformities than raphid diatoms (Pandey et al., 2017). Frustules of raphid diatoms bear an especially long continuous opening (raphe), which may provide a gateway for particular toxicants to enter the cells and cause deformities. In the present study, Type 1 (frustule outline) deformities are prevalent at all three examined waste sites and Type 2 (striae) deformities were found only at riverine waste sites (RWS), but individual diatoms with both types of deformities were not observed. Past studies indicate that the occurrence of more than one type of deformity in diatom samples is a strong indicator of metal contamination (Pandey et al., 2015; Pandey et al., 2016; Pandey et al., 2017), which adds evidence of metal contamination at RWS, although the 0.4% occurrence of deformities at this site is below the >1% that is considered a significant indicator of metal contamination (Morin et al., 2016; Pandey et al., 2017).
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.