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Lead is a toxic heavy metal which affects almost every organ in the body and specially the nervous system. Lead is used in industries and other commercial establishments regularly, due to its properties like softness, malleability, ductility, poor conductibility and resistance to corrosion. Hence, it is important to understand the toxicity status and also the adverse effect on aquatic fauna which is a major cause of concern for their gradual decline.
Being able to live a dual life, amphibians are more exposed to these heavy metal elements. The present study aims at examining the lethal and sub-lethal effect of Lead Nitrate [Pb(NO3)2] on the larvae of Indian cricket frog (Fejervarya limnocharis). Gosner 26-30 stages of tadpoles were used for the study. Tadpoles were treated with five concentrations of Pb(NO3)2, viz., 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 observed routinely. The treatments showed significant mortality. 100% lethality of the larvae prior to metamorphosis were recorded in groups treated with higher concentrations of Pb(NO3)2. Genotoxicity tests were carried out using micronucleus test in vivo. The occurrence of micronucleus was found to be statistically significant in erythrocytic cells with increasing treatment concentrations. Thus, it can be stated that environment relevant concentration of Pb(NO3)2 exposure can have deleterious effects on population and genetic diversity of Fejervarya limnocharis.
Keywords: Lead Nitrate, Fejervarya limnocharis, metamorphosis, genotoxicity, microncleus
Lead is globally considered as one of the poisonous and a ubiquitous environmental toxicant. Due to non-biodegradable nature and high persistence of lead in the environment and continuous use, its level is raising gradually, posing a serious threat for both human and animal (Wani, et. al., 2015). Lead adversely affects multiple organs including urinary, nervous, cardiovascular, skeletal, immune, gastrointestinal and reproductive system (Koh, et. al., 2015). It severely affects the nervous system and changes testicular functions in human beings and in the wildlife (Wani, et. al., 2015; Assi, et. al., 2016). Considered as a probable human carcinogen, lead exposure has been associated with cancers of the brain, stomach, kidney, lung, 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).
Gradual decline in the amphibian population is a critical issue for the researchers around the globe. Amphibians are one of the best bio-indicators of an environment health. Being terrestrial as well as aquatic, the amphibians play an important role to sustain the ecology of both the ecosystems. As they lead a dual mode of life, they are more exposed to environmental alterations as compared to other organisms. Excessive use of heavy metals in the course of industrialization and modernization has affected the amphibian population to a considerable degree.
Genetic toxicity is of vital importance because of the fact that the consequences of genetic defects have the potential to pass on to the next generation and hence affecting an entire population. Genotoxicity data is important because environmental contaminants can lead to a reduction in genetic diversity resulting from strong selection for chemical tolerance or population decline leading to genetic bottleneck and drift. In such populations, disease outbreaks can quickly assume the form of epidemic which may threaten the entire population with the possibility of extinction. Therefore, genotoxicity data is important to identify 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 recent declines, a situation far worse than that reported for mammals or birds (Stuart et al., 2004). Amphibian species and population declines are likely the result of a multitude of causes including habitat destruction, infectious disease, outbreaks, altered host parasite interactions, introduction of alien species and xenobiotic exposure (Davidson and Knapp,2007; Relyea and Diecks, 2008; Relyea, 2009). A growing number of laboratories across the world are evaluating the ecological impact of xenobiotics and nano-particles of heavy metals on amphibian at species and community level. One of the best ways to estimate risk assessment of heavy metal compounds on amphibians is to use biological tests in vivo. The present study was undertaken to examine the effect of Pb(NO3)2 on the larvae of Indian cricket frog (Fejerverya limnocharis).
Tadpoles of F. limnocharis were collected from perennial ponds nearby the study station, not contaminated by any source of pesticide and other anthropogenic exposure. The tadpoles were then acclimatized to the laboratory conditions in aged well water in polypropylene containers. Subsequently they were screened to identify and separate tadpoles belonging to Gosner 26-30 stages (Gosner, 1960). This period corresponds to intense hematopoiesis with active cell division in the circulating blood. The remaining larvae were released at the site of selection. The experiments were performed at 26±1ºC and 12 h light and dark cycles. The tadpoles were fed with crushed fish food pellets (Amrit Feeds, Kolkata, India) ad libitum. For all of experiments, animal care was in accordance with institutional ethical guidelines
Larval rearing and toxicity testing were done by following standard toxicology protocols as described elsewhere (Relyea & Mills, 2001; Reylea, 2004). Tadpoles were reared in aged well water. Stage 26 (Gosner, 1960) larvae were put in separate experimental tubs using randomized block designs. After specified time intervals, larval growth and mortality were determined. Besides, the time to metamorphosis and activity pattern were recorded in regular basis. `
Larval survival experiments were performed in polypropylene tubs (43cm×27cm×15cm) containing 2 liters of aged well water. Each tub contained 10 larvae. Chemical treatments consisted of a negative control (without any treatment), five different concentration of Pb(NO3)2, namely (12.5 µg/L, 25 µg/L , 50 µg/L, 100 µg/L and 200 µg/L).
The tub water was changed every alternate day and dosages were again applied in to the respective tubs. Each day the numbers of surviving tadpoles were counted and any dead tadpoles if we found, then were removed from the tub very carefully. Then the metamorphosis was monitored each day and any metamorphosed tadpole was removed from the tub. The experiment was continued for 35-40 days till all the tadpoles in the control tubs were fully metamorphosed.
Genotoxicity tests were carried out using micronucleus test in vivo (Jaylet Test) as described by Jaylet (1986) and described elsewhere. In brief, following appropriate time of treatment, tadpoles were anesthetized and the 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 min, and air dried. The following day, the slides were stained with Giemsa solution. The micronuclei frequency was determined in 1,000 erythrocytes from each tadpole using 1,000x magnification. Coded and randomized slides were scored blind by a single observer. The frequency of micro nucleated cells was expressed per 2,000 cells.
The survival times of tadpoles exposed to different concentrations of Pb(NO3)2 was compared using Kaplan-Meier product limit estimate. Determination of LC50 values was done using probit analysis. ANOVA was used to analyse all data related to time to metamorphosis, change in body weight, and the micronucleus frequency at different concentration levels. The analyses were performed using SPSS 18.0 statistical software at 95% confidence interval (Cl). Variances were considered to be significant at p value less than 0.05.
Pb(NO3)2 treatment on F. limnocharis tadpoles with increasing concentrations caused increased mortality which was both concentration and time dependent (Fig. 1). The tadpole survival pattern till 13th day (the day on which first metamorphosis was observed in 12.5 µg/L treatment concentration) was studied. The higher treatment concentrations 50 µg/L, 100 µg/L and 200 µg/L caused 100% mortality at 4th, 8th, and 13th day respectively. It could be effortlessly observed from the following line graph that the higher concentration of Pb(NO3)2 causes high mortality. The lower treatment concentrations 12.5 µg/L and 25 µg/L showed 96.6 % and 86.6 % survival on the 13th day of Pb(NO3)2 exposure.
The LC50 values of Pb(NO3)2 was determined between 24 to 96-h. Exposure to 12.5 µg/L and 25 µg/L of Pb(NO3)2 did not cause any lethality to the tadpoles up to 96 h of treatment. Therefore, 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. The 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 till metamorphosis. However, those exposed to lower concentrations metamorphosed early in a concentration-dependent manner. The average metamorphosis time in the groups receiving 12.5 µg/L and 25 µg/L Pb(NO3)2 treatment concentration was determined to be highly significant (p < 0.01) when compared to control group (Fig 2). Tadpoles in the control group took an average time of 20.03 ± 2.82 days for metamorphosis. The percentage of survival up to metamorphosis were 48.27%, 34.48% and 0% for groups exposed 12.5 µg/L, 25 µg/L and other higher concentrations of Pb(NO3)2 respectively. The average 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. On visual inspection, there were apparently no major abnormalities in the limb development of the metamorphosed individuals in any of the exposure groups.
Lead Nitrate treatment induced micronuclei in the erythrocytes of F. limnocharis tadpoles. Genotoxicity study was carried out for 48 hours. Scoring of Micronucleus at 48-h (r = 0.927; p < 0.05) showed significant micronucleus induction in 25 µg/L and above as compared to the untreated control. In the Post hoc analysis, 12.5 µg/L Pb(NO3)2¬ did not show any statistical significant formation of micronucleus as compared to the control groups. A positive control cyclophosphamide (2mg/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 micronucleus was observed in the higher treatment groups. MN induction was found to increase significantly as treatment concentration increases.
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 goods (Grant., 2010). Lead use has continued to grow, and in recent times has risen from five million tons per annum in 1970 to approximately 11.5 million tons in 2017 (ILZSG, 2018). Lead and lead compounds are generally toxic pollutants producing great risk to environment, human and other vertebrates (Chiesa, et. al., 2006). Lead salts and organic lead compounds are ecotoxicologically most harmful. EPA and International Agency for Research on Cancer has assigned lead a weight-of-evidence carcinogen classification of B2, probable human carcinogen, based on inadequate information in human and sufficient data in animals. The biochemical and molecular mechanisms of action of lead remain unclear, there are some studies that point out indirect mechanisms of genotoxicity such as inhibition of DNA repair or production of free radicals (García-Lestón, et. al., 2010). In the present study, LC50 value for Pb(NO3)2 for 24h, 48h, 72h and 96h were calculated to be 812.34 µg/L /L, 300.82 µg/L /L, 178.80 µg/L and 104.38 µg/L respectively. LC50 value was calculated using probit analysis in SPSS 18.0 ®. The LC50 value estimated for F. limnocharis may be helpful in studies related to environmental impact assessment of lead in the context to amphibian population decline. Lead could cause a decrease in those populations due to its lethal and sub-lethal effects. It was reported in an in-situ study carried out in wetlands located along the Merri Creek corridor in Victoria, south-eastern Australia, heavy metal contamination of copper, nickel, lead, zinc, cadmium and mercury was negatively correlated to the anuran species richness (Ficken and Byrne, 2012). The present survival study was carried out till first metamorphosis observed at 13th day of the exposure duration. In the survival study 100% mortality was observed in the higher treatment concentrations of 100 µg/L and 200 µg/L respectively and 3.33%, 80% and 86.6% survivability was observed at 50 µg/L, 25 µg/L and 12.5 µg/L respectively at 13th day of Pb(NO3)2 exposure. Thus, anuran survival was found to be negatively correlated to the increasing treatment concentration of Pb(NO3)2 similar to the report of Ficken and Byrne, 2012. According to World Health Organisation and Bureau of Indian standards, guidelines values for lead used in water treatment or lead in contact with drinking water that are of health significance in drinking-water was 10 µg/L (BIS, 2010; WHO, 2017). Therefore, it is evident that Pd at environmentally relevant concentrations can cause significant mortality in F. limnocharis tadpoles, which may have implications in amphibian population decline.
Amphibian larva exhibits developmental plasticity during growth period in response to the changing environment (Johansson et al., 2010). The strategy helps to survive in stressful condition. Environmental stressors have been reported to decline population number and shorten or delay time to metamorphosis in amphibians (Relyea, 2007; Ficken and Byrne, 2012). In the present study it was observed that the higher treatment concentrations of Pb(NO3)2 to F. limnocharis tadpoles causes death before metamorphosis. In a previous report, similar results have been reported in R. pipiens (Chen et. al., 2009). However, in contrast to their findings, in the present study, the group exposed to the lower concentrations (12.5 and 25 µg/L) of Pb(NO3)2 shows shortening of metamorphosis time significantly in the tadpoles of F. limnocharis compared to the negatively treated group. Therefore, it can be stated that exposure of Pb(NO3)2 have negative impact to the fitness of F. limnocharis.
Erythrocytes in amphibians are nucleated and undergo cell division in the circulation especially during the larval stages. Micronucleus formation is either clastogenic (chromosomal breakage) or aneugenic (loss of whole chromosome due to spindle fiber breakage) where a chromosome fragment or whole chromosome lags behind during anaphase phase. Micronucleus assay is widely practiced biomonitoring tool used worldwide to study sensitivity of aquatic organisms to genotoxic agents. Amphibian genotoxicity testing has been successfully used as biomarker for DNA damage related to environmental pollution and pesticide contamination (Maselli et. al., 2010). Therefore these cells are suitable for micronucleus detection which can be readily counted in blood smears (Giri et. al., 2011). The genotoxicity evaluations were made at 48 h of the exposure in contrast to the traditional 24 h exposure period for other animals especially in mammals. This is because, in previous reports it has been shown that the ideal time for assessment of genotoxicity in tadpole is 48 h as it shows the maximum frequency of micronucleus in the time response study (Giri et al., 2012; Yadav et al., 2013).
In the present study it was observed that MN induction increases in dose dependent manner at 48 h of exposure duration when treated with different concentration of Pb(NO3)2. However, in contrast to our finding a report published previously stated MN study showed negative results when Chinese Hamster ovary were treated with Pb(NO3)2 (Lin et al., 1994; García-Lestón, et. al., 2010). While other reports show similar findings of MN occurrence due to lead exposure, Poma et al. (2003) found that lead acetate induced MN in a dose dependent manner when evaluated chromosomal damage induced in human melanoma cells (B-Mel). Montaldi et al. (1987), evaluated the effects of lead sulphate and lead chromate on the induction of MN in human lymphocytes when similar results were observed. Thier et al. (2003) and Bonacker et al. (2005) studied the genotoxic effects of inorganic lead salts in V79 Chinese hamster fibroblasts by means of MN test. They observed that lead chloride and lead acetate induced MN in a dose dependent manner. Therefore, over all it can be stated that lead causes genotoxicity to the tadpoles of F. limnocharis and it is also suggested that more experiments should be carried out to study the effects of lead on aquatic animals from different aspects to completely understand the underlying process of interaction between environmental toxicant and interacted aquatic animals.
In summary, the present finding puts light on the toxic and genotoxic effects of Pb(NO3)2 in F. limnocharis tadpoles. Effects of Pb(NO3)2 reveals the long-term fitness consequences to the population of amphibians as a whole. The findings of genotoxicity study shows that Pb(NO3)2 is genotoxic to F. limnocharis. This could result in adverse population effects in the long run resulting in deleterious effects such as loss of genetic diversity in the population and hence exert negative impact on the population structure and hence lead to population decline. However, more studies on genotoxicity assessments in amphibians are suggested. Besides the present study have significant methodological implications. More studies using different concentrations of Pb(NO3)2 is necessary for full understanding of the interactions of the chemical and other amphibian population.
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