Behavioral Biomarker Responses of Filopaludina bengalensis to Acute Copper Toxicity

As heavy metals are bioaccumulative and toxic in nature, increasing heavy metal levels in the environment is of great concern.The present study was aimed to assess the acute toxicity effects of copper on some behavioral attributes of Filopaludina bengalensis (locally known as ‘Genri’), a freshwater gastropod, under laboratory condition. Exposure to copper (0.50-2.0 ppm) for 96 h resulted in significant mortality. Recorded LC50 value at 96h was 0.586 ppm. The aggregation tendency, crawling activity and mucus secretion were observed at different exposure period upon acute exposure of copper (0.586 ppm). With the advancement of exposure period, gradual deaggregation was observed in snails of copper treated sets. There was statistically significant difference (P <0.01) in aggregation response and foot protrusion response at different span of exposure in copper treated groups. In copper treated sets, a substantial increase (P<0.01) in mucus secretion (%) was evident with time. Foot protrusion and movement was inhibited in copper treated groups. All three responses in the copper treated groups were significantly different (P <0.01) from that of control groups. The behavioral responses observed in this study of Filopaludina bengalensis could be used as biomarkers for copper contamination in any aquatic ecosystems. CONTACT Pratyush Ghosh pratyushghosh60@gmail.com Department of Zoology,Chandernagore College, Chandannagar, Hooghly-712136, W.B, India. © 2021 The Author(s). Published by Enviro Research Publishers. This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY). Doi: http://dx.doi.org/10.12944/CWE.16.1.23 Article History Received: 19 August 2020 Accepted: 08 March 2021


Introduction
Indiscriminate water pollution due to urbanization, industrialization and modern agricultural practices alter physical, chemical and biochemical characteristics of water bodies (Indra and Shivaji, 2006). Heavy metal pollution in freshwater ecosystem is of great environmental concern in these days because of bioaccumulative and non-biodegradable nature of the heavy metals (Walker et al., 2006). The natural sources of heavy metals include weathering of rocks and volcanic eruptions, while various industrial activities and mining are their anthropogenic sources (Ali et al., 2019). The concentration of metals in aquatic organisms appear to have been many times greater than the levels that actually exist in the ecosystem (Laws, 2000).Metals become concentrated more at higher trophic levels, presumably due to biomagnification (Wyn et al., 2007). Some heavy metals are essential for life but they could impart toxic effects through bioaccumulation (Gobi et al., 2018). Copper is an essential component of different enzymes but toxic for all forms of life at its excessive level (Gawad, 2018).
A variety of methods have been developed to assess the hazard and toxicity of heavy metals to organisms such as acute toxicity test, chronic toxicity test etc. (Singh and Zahara, 2017). Acute toxicity is the severe or adverse biological effects, which occur within a short period of time after a short term exposure to toxic chemicals (Kopruccu et al., 2006). LC 50 or median lethal concentration, a statistically derived concentration, is expected to cause death in 50% of the test species over a given exposure period (Qian et al.,2020). Higher LC 50 value indicates less toxic because greater concentrations of the concerned toxicants are required to produce 50% mortality in organisms. Molluscs, specially gastropods and bivalves have been long regarded as promising bioindicators and biomonitoring subjects. They are easily available, easy to grow and extremely tolerant to many pollutants and can also accumulate heavy metals (Kader et al., 2016). Filopaludina bengalensis is a filter feeding benthic gastropod, locally known as 'Genri' (Subba Rao and Dey,1991). They are indiscriminately collected from natural habitats and used as poultry food, cheap proteinaceous food item for rural tribal population in India (Baby et al., 2010).
Generally, the biomarkers are xenobiotically induced measurable physiological, biochemical, behavioral and other alternations in a biological system or samples. Occurrence of biomarkers ranges from subcellular level to whole organism and ecosystem level (Hamza-Chaffai, 2014). Behavior is designed to ensure maximal fitness as it is highly coordinated, structured and predictable sequence of activities rather than a random process (Halappa and David, 2009). Alternations in normal behavioral repertoire can be resulted in reduced fitness and survival of individual organisms, which in turn can elicit undesirable changes in population (Brewer et al., 2001). Behavioral alternations in animals are very sensitive endpoint in ecotoxicology; they can serve as a valuable tool to evaluate effects of exposure to various environmental stressors (Deyashi et al., 2019;Alonso and Camargo, 2011). Copper contamination in freshwater aquatic habitat is very common in Gangetic plains of India. Quest for effective bioindicator species to detect and monitor heavy metal contamination in freshwater systems is the need of the hour. Keeping this in mind, the present study was undertaken with objectives to determine the LC 50 value and to evaluate the impact of acute copper intoxication on some behavioral biomarker attributes of Filopaludina bengalensis. This study may serve as a reference for the assessment of the ecotoxicity of copper in many other species. This research could also help to understand the health risks of copper toxicity.

Snail collection
The snails Filopaludina bengalensis were collected in the early morning from an unpolluted fish culture pond located near Chandannagar, Hooghly, India (22°87'N and 88°38'E) during June, 2019 ( Fig. 1). A total 1010 Filopaludina bengalensis were collected by handpicking. They were then transported to laboratory. The shell length of each snail was measured using the divider and meter ruler. Their wet weight was also taken. The specimen selected for the experiment had an average 2.7 cm (range 2.2 to 2.91 cm) shell length and average 2.03 g wet weight (range 1.31 to 2.47 g). They were acclimatized in 5 L glass aquaria for 7 days to laboratory regimes before experimentation. During this acclimatization period, they were fed with blanched lettuce, papaya leaves and libitum plant leaves.

Bioassay
In order to determine the concentration of heavy metals to be used in the actual experiment, a rough range finding test was carried out for a span of 96 h. The standard stock solutions (100 mg/L) were prepared with deionized water from reagent analytical grade metallic salts of copper. Copper sulfate pentahydrate (CuSO 4 , 5H 2 O) was used. Graded series of concentrations were prepared from stock solutions based on APHA (1998) guidelines. Six test concentrations -(0.5, 0.75, 1.0, 1.25, 1.5 and 2.0 ppm) were used for the determination of the LC 50 (median lethal concentration) value. The whole experiment was performed at room temperature 27-30°C with photoperiod ~13 h light and ~11 h darkness. Physicochemical parameters of the diluent medium pH, TDS and temperature were determined using portable meters and alkalinity and DO were measured following standard methods of APHA (2012). The mean value and the standard deviation of the parameters for water quality were: Alkalinity: 118.7±15.6 mg/L, DO: 6.93±0.21 mg/L, TDS: 663.5±42.8 mg/L, pH: 7.2±0.2. Acute toxicity experiment of Cu was performed for 96h period using adult Filopaludina bengalensis. Group of 20 adult snails were placed in 5L glass aquaria containing appropriate solution. There was a control group with only dechlorinated tap water. The rest of the six groups were exposed to series of metallic salt solution. There were 3 replicates of both control and heavy metal treated groups.A total of 20 snails were used per treatment/concentration including control.Mortality was recorded at the end of 24, 48, 72 and 96 hours of treatment during the toxicity test.The dead snails were removed quickly.
The inability of the animal to respond normally to gentle physical stimulation on foot with blunt needle was used as the criteria for determination of death/ mortality. Further confirmation of death was done by placing the animal on glass Petridis for few minutes. If it did not move, it was considered dead.Snails were not fed during the toxicity test.

Study of the Behavioral Responses
Four experimental sets : Set-A (exposed up to 24 h), Set-B (exposed up to 48 h), Set-C (exposed up to 72 h) and Set-D (exposed up to 96 h) each containing 20 snails were exposed to 0.586 ppm (96 h LC 50 ) copper solution. Equal number of individuals exposed to dechlorinated tap water was maintained as control set. Three replicates were set up for each of the sets (both treatment groups and control). Behavioral responses of Filopaludina bengalensis of both the treated and control groups of different exposure period (24 hours, 48 hours, 72 hours, and 96 hours) were recorded. Three types of behavioral responses were observed and recorded here-i) Aggregation and clumping tendency ii) Mucus secretion and iii) Foot protrusion and foot movement. These behavioral responses were documented at 6 h interval in both control and exposed groups. All the results are expressed in percentage.

Statistical Analysis
Finney's Probit Analysis LC 50 determination method was used to measure LC 50 values, 95% confidence limits, slopes and intercepts of 24 hours, 48 hours, 72 hours, and 96 hours (Finney 1971). As none of the three behavioral measures did not confirm to normality assumption (Kolmogorov -Smirnov test, P < 0.05), for multiple comparison Kruskal-Wallis test and Mann-Whitney U test was done with SPSS version18 software. The results obtained from the behavioral study were represented as mean ± SE (Standard Error).

Results
Acute toxicity of copper revealed that mortality is proportional to the concentration of heavy metal directly. No mortality was evidenced in control. All snails survived in the control throughout the experiment period.

Discussion
The acute toxicity of copper in Filopaludina bengalensis has been reported in a few studies. Direct comparisons of the toxicity values obtained in this study with those in the literature is cumbersome, since they differed between closely related species and also between the same species (Okocha and Adedeji, 2011), primarily due to differences in physicochemical characteristics of the test water and the age of the tested organisms (Wang et al., 2010). The present study reveals that rate of mortality increased as concentration of copper increased and mortality rate rises as the period of exposure increased. These findings also indicate the impact of copper was dependent on dose and exposure time. Kamble and Kamble (2012) showed that 96h-LC 50 of copper to Bellamya bengalensis was 0.56 ppm which was quite close to the findings of present investigation.In comparison to other gastropods, the 96 h LC 50 value of Copper in Filopaludina bengalensis as observed in the present study is shown in Table 2.
In this study, it was observed that the behavioral responses of the snails of the treated sets at 24 h, Filopaludina bengalensis has evolved a welldeveloped muscular foot which protrudes occasionally through the opercular aperture to fulfill diverse purposes like locomotion, food procurement, reproduction etc. Reduction in foot protrusion and movement as observed in this study was also observed in some other studies.Exposure to copper hydroxide has shown to have an effect on movement of Leidyulla florida (Capinera and Dickens, 2016). Moreover, withdrawal of foot inside the shell can help them to tightly close their operculum (Kamble andKamble, 2014).
When creeping on substratum, snails naturally secrete watery thin mucus by foot. In the aquatic snail Filopaludina bengalensis, such mucus secretion is also observed. Intoxication of metals caused increased quantity of mucus secretion in gastropods (Yasmeen and Begum, 2015). One of the early responses shown by various gastropods to environmental stressors is increased mucus secretion (Triebskorn et al., 1998). Thick and large amount of mucus was secreted by the foot, while gills secreted somewhat more whitish mucus that comes out of the mouth and in treated sets, its quantity also increased as time went on (Deshmane, 2012). It may be to inhibit metal absorptions into the organism (Lobel, 1981). Mucus acts as a physicochemical barrier of animal against environmental toxins. Extruded mucus forms a kind of protective barrier that helps to keep the epithelia of skin or digestive tract from the direct contact of toxin (Ebenso, 2004;Ebenso et al., 2005). A substantial increase in mucus secretion was observed in Perna viridis upon treatment with 0.5 mg/L copper by Sze and Lee (1995). Heavy mucus secretion in Viviparus bengalensis was observed by Muley and Mane (1988) upon exposure to mercuric salt. Kamble and Kamble (2014) also recorded profuse mucus secretion, reduced foot and tentacular movement as a response to intoxication of copper sulphate. Mollusc also faced the higher risk of dehydration as extruded mucus having higher water content. They lose sufficient energy also, which may leave them motionless (Cottrell et al., 1993;Ebenso, 2004).
An organism's behavior is the final, integrated outcome of various physiological and biochemical processes. Therefore behavioral changes could represent higher organization level of biomarker. A behavioral parameter is more comprehensive and informative than a biochemical or physiological parameter (Walker et al., 2006). The behavioral repertoire in gastropod is limited compared to vertebrates; yet extensive and variable enough to be applied in ecotoxicological studies. The mortality percentage and the biomarker responses observed in this study in Filopaludina bengalensis may be used as indicative parameters for assessing the toxicity of copper in aquatic ecosystems (Doving, 1992).

Conclusion
This study revealed that acute exposure to copper has a major impact on aggregation, foot protrusion response, and secretion of mucus in Filopaludina bengalensis. As the exposure time increased, further deaggregation, inhibition of foot movement and strong mucus secretion were observed. This present study also indicates that the changes in behavioral attributes caused by copper in Filopaludina bengalensis at the individual or population level may be used as an effective biomarker for monitoring and management of aquatic heavy metal pollution.