Table of contentsAbstractIntroduction:Materials and methods:Results:Discussion:AbstractLead is a toxic heavy metal that affects almost all organs of the body and especially the nervous system. Lead is regularly used in industries and other commercial establishments, due to its properties such as softness, malleability, ductility, poor conductivity and corrosion resistance. It is therefore important to understand the toxicity status as well as the adverse effects on aquatic fauna, which constitute a major source of concern regarding their progressive decline. Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get the original essay Being able to live a double life, amphibians are more exposed to these heavy metal elements. The present study aims to examine the lethal and sublethal effect of lead nitrate [Pb(NO3)2] on Indian cricket frog (Fejervarya limnocharis) larvae. Gosner, 26 to 30 tadpole instars were used for the study. Tadpoles were treated with five concentrations of Pb(NO3)2, namely 12.5 µg/L, 25 µg/L, 50 µg/L, 100 µg/L and 200 µg/L. Survival and metamorphosis of treated larvae were regularly observed. The treatments showed significant mortality. 100% mortality of larvae before metamorphosis was recorded in groups treated with higher concentrations of Pb(NO3)2. Genotoxicity testing was performed using an in vivo micronucleus assay. The appearance of micronuclei was found to be statistically significant in erythrocyte cells with increasing treatment concentrations. Thus, it can be stated that an environmentally relevant concentration of exposure to Pb(NO3)2 can have deleterious effects on the population and genetic diversity of Fejervarya limnocharis.Keywords: Lead nitrate, Fejervarya limnocharis, metamorphosis , genotoxicity, microncleusIntroduction:Lead is globally considered to be a toxic and omnipresent environmental toxicant. Due to the non-biodegradable nature of lead, its high persistence in the environment and its continuous use, its level is gradually increasing, posing a serious threat to humans and animals (Wani, et. al., 2015 ). Lead has adverse effects on multiple organs, including the urinary, nervous, cardiovascular, skeletal, immune, gastrointestinal, and reproductive systems (Koh, et. al., 2015). It seriously affects the nervous system and modifies testicular functions in humans and wildlife (Wani, et. al., 2015; Assi, et. al., 2016). Considered a probable human carcinogen, lead exposure has been associated with brain, stomach, kidney, lung and meningeal cancers (Boffetta et al. 2011; Van Bemmel et al. 2011 ;) Therefore, more research is needed to understand the relationship between lead and cancer (Koh, et. al., 2015). The gradual decline of the amphibian population is a critical problem for researchers around the world. Amphibians are one of the best bioindicators of environmental health. Being terrestrial and aquatic, amphibians play an important role in maintaining the ecology of both ecosystems. As they lead a dual lifestyle, they are more exposed to environmental alterations than other organisms. Excessive use of heavy metals during industrialization and modernization has significantly affected the amphibian population. Genetic toxicity is of vital importance because the consequences of genetic defects can potentially betransmitted to the next generation and therefore affecting an entire population. Genotoxicity data are important because environmental contaminants can cause a reduction in genetic diversity resulting from strong selection for chemical tolerance or a population decline leading to bottlenecking and genetic drift. In such populations, outbreaks can quickly take the form of an epidemic that can put the entire population at risk of extinction. Therefore, genotoxicity data are important for identifying genetic diversity (Murdoch and Hebert 1994), contamination-induced natural selection (Peles et al. 2003), and increased mutation rates (Somers et al. 2002). According to the first global assessment of the status of amphibian species, more than 40% of the world's amphibian species have recently experienced declines, a situation far worse than that reported for mammals or birds (Stuart et al ., 2004). The decline of amphibian species and populations is likely the result of a multitude of causes, including habitat destruction, infectious diseases, epidemics, altered host-parasite interactions, introduced species exotics and exposure to xenobiotics (Davidson and Knapp, 2007; Relyea and Diecks, 2008; Relyea, 2009). A growing number of laboratories around the world are assessing the ecological impact of xenobiotics and heavy metal nanoparticles on amphibians at the species and community level. One of the best ways to assess the risks posed by heavy metal compounds to amphibians is through the use of in vivo bioassays. The present study was undertaken to examine the effect of Pb(NO3)2 on larvae of Indian cricket frog (Fejerverya limnocharis).Materials and methods: Tadpoles of F. limnocharis were collected from perennial ponds near the study station, not contaminated by any source of pesticides and other anthropogenic exposures. Tadpoles were then acclimated to laboratory conditions in aged well water in polypropylene containers. Subsequently, they were examined to identify and separate tadpoles belonging to Gosner stages 26-30 (Gosner, 1960). This period corresponds to intense hematopoiesis with active cell division in the circulating blood. The remaining larvae were released at the selection site. Experiments were performed at 26 ± 1 °C and with 12 h light and dark cycles. Tadpoles were fed crushed fish food pellets ad libitum (Amrit Feeds, Kolkata, India). For all experiments, animal care was in accordance with institutional ethical guidelines. Larval rearing and toxicity testing was carried out following standard toxicological protocols described elsewhere (Relyea & Mills, 2001; Reylea, 2004). Tadpoles were raised in aged well water. Stage 26 larvae (Gosner, 1960) were placed in separate experimental tanks using randomized block designs. After specified time intervals, larval growth and mortality were determined. Additionally, metamorphosis time and activity pattern were recorded regularly. “Larval survival experiments were carried out in polypropylene tanks (43 cm × 27 cm × 15 cm) containing 2 liters of aged well water. Each tank contained 10 larvae. The chemical treatments consisted of a negative control (without any treatment), five different concentrations of Pb(NO3)2, namely (12.5 µg/L, 25 µg/L, 50 µg/L, 100 µg/L and 200 µg/L). The water in the tank was changed every two days and the doses were reapplied to the respective tanks. Every day, the number ofSurviving tadpoles were counted and any dead tadpoles, if found, were then very carefully removed from the tub. Thereafter, metamorphosis was monitored daily and any metamorphosed tadpoles were removed from the tub. The experiment was continued for 35 to 40 days until all the tadpoles in the control tanks were completely metamorphosed. Genotoxicity testing was performed using an in vivo micronucleus assay (Jaylet assay) as described by Jaylet (1986) and described elsewhere. Briefly, after an appropriate treatment time, the tadpoles were anesthetized and the blood samples were collected by cardiac puncture. Three blood smears for each animal were immediately prepared on clean slides, fixed in absolute methanol for 3 min, and air dried. The next day, the slides were stained with Giemsa solution. The frequency of micronuclei was determined in 1,000 erythrocytes from each tadpole using 1,000x magnification. Coded and randomized slides were scored blindly by a single observer. The frequency of micronucleated cells was expressed per 2,000 cells. Survival times of tadpoles exposed to different concentrations of Pb(NO3)2 were compared using Kaplan-Meier product limit estimation. Determination of LC50 values ​​was carried out using probit analysis. ANOVA was used to analyze all data related to time taken for metamorphosis, change in body weight, and frequency of micronuclei at different concentration levels. Analyzes were performed using SPSS 18.0 statistical software at a 95% confidence interval (Cl). Variances were considered significant at a p-value less than 0.05. Results: Pb(NO3)2 treatment of F. limnocharis tadpoles with increasing concentrations resulted in increased mortality that depended on both concentration and time (Fig. 1). The survival profile of tadpoles until day 13 (the day the first metamorphosis was observed at a treatment concentration of 12.5 µg/L) was studied. The higher treatment concentrations, 50 µg/L, 100 µg/L, and 200 µg/L, caused 100% mortality at days 4, 8, and 13, respectively. It can be easily observed from the following line graph that the higher concentration of Pb(NO3)2 results in high mortality. The lower treatment concentrations, 12.5 µg/L and 25 µg/L, showed 96.6% and 86.6% survival at day 13 of Pb(NO3)2 exposure. LC50 values ​​for Pb(NO3)2 were determined between 24 and 96 hours. Exposure to 12.5 µg/L and 25 µg/L Pb(NO3)2 did not cause any lethality in tadpoles up to 96 h of treatment. Therefore, the LC50 values ​​for 24 h, 48 h, 72 h and 96 h of exposure were 812.34 µg/L, 300.82 µg/L, 178.8 µg/L and 104.38 µg/L, respectively. L. LC50 values ​​decreased over time (r = 0.986, p < 0.05). Tadpoles exposed to Pb(NO3)2 resulted in accelerated metamorphosis. Tadpoles exposed to higher concentrations of Pb(NO3)2 (50 µg/L, 100 µg/L and 200 µg/L) did not survive to metamorphosis. However, those exposed to lower concentrations metamorphosed early, in a concentration-dependent manner. The mean time to metamorphosis in the groups receiving Pb(NO3)2 treatment concentration of 12.5 µg/L and 25 µg/L was determined to be highly significant (p < 0.01) compared to the control group. (Fig. 2). Tadpoles in the control group took a mean time of 20.03 ± 2.82 days to metamorphose. The survival percentages until metamorphosis were 48.27%, 34.48% and 0% for the groups exposed to 12.5 µg/L, 25 µg/L and other higher concentrations of Pb, respectively. (NO3)2. Body weightmean of metamorphosed individuals was significantly reduced (p < 0.05) and (p < 0.01) in the groups exposed to 12.5 µg/L and 25 µg/L of Pb(NO3), respectively. Upon visual inspection, there were apparently no major abnormalities in the limb development of metamorphosed individuals in any of the exposed groups. Lead nitrate treatment induced micronuclei in erythrocytes of F. limnocharis tadpoles. The genotoxicity study was carried out for 48 hours. Micronucleus scoring at 48 h (r = 0.927; p < 0.05) showed significant micronucleus induction at 25 µg/L and above compared to the untreated control. In post hoc analysis, 12.5 µg/L Pb(NO3)2¬ showed no statistically significant micronucleus formation compared to the control groups. A positive control, cyclophosphamide (2 mg/L), was studied for reference. The overall time effect on micronucleus induction (ANOVA) was statistically significant (F5, 90.430, p < 0.01). The highest frequency of micronuclei was observed in the higher treatment groups. It was found that MN induction increased significantly as the treatment concentration increased. Discussion: EPA regulates lead under the Clean Air Act (CAA) and has designated lead as a hazardous air pollutant (HAP). Lead is used in various fields viz. in water distribution systems, paints, fuel additives and electronic products (Grant., 2010). Lead use has continued to grow and recently increased from five million tonnes per year in 1970 to approximately 11.5 million tonnes in 2017 (ILZSG, 2018). Lead and its compounds are generally toxic pollutants that pose a significant risk to the environment, humans and other vertebrates (Chiesa, et. al., 2006). Lead salts and organic lead compounds are the most harmful from an ecotoxicological point of view. The EPA and the International Agency for Research on Cancer have assigned a weight-of-evidence classification of carcinogen, B2, probable human carcinogen, based on inadequate information in humans and sufficient data in animals. The biochemical and molecular mechanisms of action of lead remain unclear, some studies highlight indirect mechanisms of genotoxicity such as the inhibition of DNA repair or the production of free radicals (García-Lestón, et. al., 2010 ). In the present study, the LC50 values ​​for Pb(NO3)2 for 24 h, 48 h, 72 h and 96 h were calculated as 812.34 µg/L/L, 300.82 µg/L/, respectively. L, 178.80 µg/L and 104.38 µg/L. The LC50 value was calculated using probit analysis in SPSS 18.0 ®. The estimated LC50 value for F. limnocharis may be useful in studies related to the assessment of the environmental impact of lead in the context of amphibian population decline. Lead could cause a decline in these populations due to its lethal and sublethal effects. It was reported in an in situ study in wetlands along the Merri Creek corridor in Victoria, southeastern Australia, that heavy metal contamination (copper, nickel, lead, zinc , cadmium and mercury) was negatively correlated with anuran species richness. (Ficken and Byrne, 2012). The present survival study was carried out until the first metamorphosis observed on the 13th day of the exposure period. In the survival study, 100% mortality was observed at the highest treatment concentrations of 100 µg/L and 200 µg/L respectively, and survivability of 3.33%, 80% and 86. 6% was observed at 50 µg/L, 25 µg/L and 12.5 µg. /L respectively on the 13th day of exposure to Pb(NO3)2. Thus, anuran survival was found to be negatively correlated with increased.