IndexSummaryIntroduction:Materials and methods:Results:Discussion:SummaryLead is a toxic heavy metal that affects almost all organs of the body and especially the nervous system. Lead is used regularly in industries and other commercial establishments, due to its properties such as softness, malleability, ductility, low conductivity and corrosion resistance. It is therefore important to understand the state of toxicity and also the negative effects on aquatic fauna, which is a major cause of concern for their progressive decline. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essayBeing able to live a double life, amphibians are more exposed to these heavy metal elements. The present study aims to examine the lethal and sub-lethal effect of lead nitrate [Pb(NO3)2] on Indian cricket frog (Fejervarya limnocharis) larvae. 26-30 Gosner stages of tadpoles 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 the treated larvae were regularly observed. The treatments showed significant mortality. 100% lethality of larvae before metamorphosis was recorded in groups treated with higher concentrations of Pb(NO3)2. Genotoxicity testing was performed using the in vivo micronucleus test. The presence of the micronucleus was statistically significant in erythrocytic cells with increasing treatment concentrations. Therefore, it can be said that the relevant environmental concentration of Pb(NO3)2 exposure can have deleterious effects on the population and genetic diversity of Fejervarya limnocharis.Keywords: lead nitrate, Fejervarya limnocharis, metamorphosis, genotoxicity, microncleoIntroduction: the Lead is globally regarded as one of the poisonous and ubiquitous environmental toxicant. Due to the non-biodegradable nature, high persistence of lead in the environment and continuous use, its level is gradually increasing, posing a serious threat to both humans and animals (Wani, et. al., 2015). Lead adversely affects multiple organs including the urinary, nervous, cardiovascular, skeletal, immune, gastrointestinal, and reproductive systems (Koh, et. al., 2015). It severely affects the nervous system and changes 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 tumors of the brain, stomach, kidneys, lungs and meninges (Boffetta, et al. 2011; Van Bemmel, et al. 2011; Koh , et. al., 2015 ;). Therefore, more research is needed to understand the relationship between lead and cancer (Koh, et. al., 2015). The gradual decline of amphibian populations is a critical issue 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 supporting the ecology of both ecosystems. Since they lead a dual mode of life, they are more exposed to environmental alterations than other organisms. The 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 be passed on to the next generation and therefore affecting an entire population. Genotoxicity data are important because environmental contaminantsthey can lead to a reduction in genetic diversity resulting from strong selection for chemical tolerance or population decline leading to a bottleneck and genetic drift. In such populations, disease outbreaks can quickly take the form of an epidemic that can threaten the entire population with the possibility of extinction. Therefore, it is important to identify data on genotoxicity, 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 experienced recent 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 interactions with host parasites, the introduction of exotic species, and l exposure to xenobiotics (Davidson and Knapp, 2007; Relyea and Diecks, 2008; Relyea, 2009). A growing number of laboratories around the world are evaluating the ecological impact of xenobiotics and heavy metal nanoparticles on amphibians at the species and community levels. One of the best ways to estimate the risk assessment of heavy metal compounds on amphibians is to use in vivo bioassays. The present study was undertaken to examine the effect of Pb(NO3)2 on the larvae of Indian cricket frog (Fejerverya limnocharis). Materials and methods: F. limnocharis tadpoles were collected from perennial ponds close to the study station, not contaminated by any source of pesticides and other anthropogenic exposures. The tadpoles were then acclimated to laboratory conditions in well water aged in polypropylene containers. They were subsequently screened 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±1ºC and 12-h light and dark cycles. Tadpoles were fed crushed fish food pellets (Amrit Feeds, Calcutta, India) ad libitum. For all experiments, animal care was in accordance with institutional ethical guidelines. Larval rearing and toxicity testing were performed following standard toxicological protocols as 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, growth and mortality of the larvae were determined. Furthermore, the time to metamorphosis and the activity pattern were recorded regularly. “Larval survival experiments were performed in polypropylene tanks (43 cm x 27 cm x 15 cm) containing 2 liters of aged well water. Each tank contained 10 larvae. 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). was changed every other day and the dosages were applied again to the respective tanks. Every day the number of surviving tadpoles was counted and if we found dead tadpoles, they were removed from the tank very carefully. Then the metamorphosis was monitored every day and any metamorphosed tadpoles was removed from the tank. The experiment wascontinued for 35–40 days until all tadpoles in the control tanks were completely metamorphosed. Genotoxicity tests were performed using the in vivo micronucleus test (Jaylet Test) as described by Jaylet (1986) and described elsewhere. Briefly, after an appropriate treatment period, tadpoles were anesthetized and blood samples were obtained by cardiac puncture. Three blood smears for each animal were immediately prepared on clean slides, fixed in absolute methanol for 3 minutes, 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 blindly scored by a single observer. The frequency of micronucleated cells was expressed per 2,000 cells. Survival times of tadpoles exposed to different Pb(NO3)2 concentrations were compared using Kaplan-Meier product limit estimation. Determination of LC50 values ​​was performed using probit analysis. ANOVA was used to analyze all data relating to time to metamorphosis, change in body weight, and micronucleus frequency at different concentration levels. Analyzes were performed using SPSS 18.0 statistical software with a 95% confidence interval (Cl). Variances were considered significant with a p value less than 0.05. Results: Pb(NO3)2 treatment on F. limnocharis tadpoles with increasing concentrations caused an increase in mortality that depended on both concentration and time (Fig. 1). The tadpole survival pattern was studied until day 13 (the day the first metamorphosis was observed with a treatment concentration of 12.5 µg/L). The highest treatment concentrations, 50 µg/L, 100 µg/L and 200 µg/L, caused 100% mortality on days 4, 8 and 13, respectively. From the following line graph it can easily be observed that the higher concentration of Pb(NO3)2 causes high mortality. The lowest treatment concentrations, 12.5 µg/L and 25 µg/L, showed survival of 96.6% and 86.6% at day 13 of exposure to Pb(NO3)2. Pb(NO3)2 LC50 values ​​were determined between 24 and 96 hours. Exposure to 12.5 µg/L and 25 µg/L Pb(NO3)2 caused no mortality in tadpoles up to 96 hours 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. LC50 values ​​decreased in a time-dependent manner (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 transformed early in a concentration-dependent manner. The mean time to metamorphosis in the groups that received a treatment concentration of 12.5 µg/L and 25 µg/L of Pb(NO3)2 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 rate to metamorphosis was 48.27%, 34.48% and 0% for the groups exposed to 12.5 µg/L, 25 µg/L and other higher concentrations of Pb(NO3), respectively. 2. The mean body weight of the 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)2, respectively. Upon visual inspection, there were apparently no major abnormalities in limb developmentmetamorphosed individuals in none of the exposure groups. Lead nitrate treatment induced micronuclei in the erythrocytes of F. limnocharis tadpoles. The genotoxicity study was conducted for 48 hours. The micronucleus score at 48 hours (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 micronuclei formation compared to control groups. A positive control of cyclophosphamide (2 mg/l) was studied as a 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 highest treatment groups. MN induction was found to increase significantly with increasing treatment concentration. Discussion: The 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). The use of lead has continued to grow and has recently increased from five million tonnes per year in 1970 to approximately 11.5 million tonnes in 2017 (ILZSG, 2018). Lead and lead compounds are generally toxic pollutants that pose great risks to the environment, humans and other vertebrates (Chiesa, et. al., 2006). Lead salts and organic lead compounds are ecotoxicologically more harmful. The EPA and the International Agency for Research on Cancer have assigned Leader a weight-of-evidence carcinogenic classification of 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, there are some studies that 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 value for Pb(NO3)2 for 24 hours, 48 ​​hours, 72 hours and 96 hours was calculated to be 812.34 µg/L /L, 300.82 µg/L /L, 178, respectively 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 evaluating the environmental impact of lead in the context of amphibian population decline. Lead could cause a decline in those populations due to its lethal and sub-lethal effects. It was reported in an in situ study conducted in wetlands located along the Merri Creek corridor in Victoria, south-eastern Australia, that heavy metal contamination of copper, nickel, lead, zinc, cadmium and mercury was negatively correlated to the richness of anuran species. (Ficken and Byrne, 2012). The present survival study was conducted until the first metamorphosis observed on day 13 of the exposure duration. In the survival study, 100% mortality was observed at the highest treatment concentrations of 100 µg/L and 200 µg/L, respectively, and survival 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. Therefore, anuran survival was found to be negatively related to the increase in Pb(NO3)2 concentration during treatment, similar to the report of Ficken and Byrne, 2012. According to the World Health Organization and the Bureau of Indian Standards, guideline values ​​for lead used in water treatments or lead in contact.