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Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles
Ecotoxicology and Environmental Safety 191 (2020) 110209
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Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles
T
Palas Samantaa,1, Sandipan Palb,1, Aloke Kumar Mukherjeec, Apurba Ratan Ghoshd,∗ a
Department of Environmental Science, Sukanta Mahavidyalaya, Dhupguri, West Bengal, India Department of Environmental Science, Aghorekamini Prakashchandra Mahavidyalaya, India c P.G. Department of Conservation Biology, Durgapur Government College, India d Ecotoxicology Lab, Department of Environmental Science, The University of Burdwan, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arsenic Chromium Almix herbicide Euphlyctis cyanophlyctis Alkaline phosphatase Glutathione S-transferase LC50
Heavy metals and herbicide are gaining serious environmental concern in aquatic toxicology due to its adverse effects on aquatic organisms especially amphibians. Accordingly, present study first time evaluated the acute toxicity of two heavy metals [arsenic (As3+) and chromium (Cr6+)] and a herbicide (Almix) to Indian skittering frog tadpole, Euphlyctis cyanophlyctis. The LC50 values of As, Cr and Almix for 24, 48, 72 and 96 h were 73.58, 56.31, 43.58 and 32.58 mg L−1; 326.68, 224.31, 171.92 and 141.99 mg L−1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L−1, respectively. It also revealed the concentration- and time-dependent increased mortality rate under these toxicants. The safety concentrations (SC) of As, Cr and Almix to tadpoles were 3.26, 14.20 and 95.52 mg L−1, respectively. The findings disclosed that As is highly toxic to E. cyanophlyctis than Cr and Almix. Alkaline phosphatase (ALP) activity showed varied responses to exposed chemicals. In particularly, ALP activity reduced significantly for Cr treatment. Glutathione S-transferase (GST) activity in E. cyanophlyctis was significantly inhibited by As treatment (p < 0.05); however, GST activity was remain unchanged for Cr and Almix (p > 0.05). The As toxicity correlates positively with GST inhibition (r = 0.779, p < 0.01); contrarily, Cr and Almix revealed negative correlation with GST induction (r = −0.461 and −0.19, respectively; p > 0.05). This result indicated that GST play a crucial role for regulating the tadpole mortality and intoxication by As, Cr and Almix. Overall, our findings demonstrate the different levels of toxic sensitivity (adverse effects) under different toxicants on E. cyanophlyctis tadpoles. Finally, the present findings could be used as baseline information of toxicosis for metalloid, heavy metal and herbicide exposures in wild frog populations.
1. Introduction According to the International Union for Conservation of Nature (IUCN) 32.5% amphibian species were vulnerable, endangered, or critically endangered; 7.4% species were critically endangered and 43% species were experiencing the population decline (IUCN, 2004; Gabor et al., 2019). Such diminution of amphibian population is mainly due to destruction and loss of habitats, like ponds, lakes, wetlands and rivers (Greulich and Pflugmacher, 2004; Li et al., 2010). Additionally, chemical contamination mainly by heavy metals and pesticides from industrial and agricultural practices also threaten the amphibian life (Sai et al., 2015). Exposure to this contaminated water, the amphibians especially can absorb these contaminants directly from water via diffusion across the skin, gonads, and various tissues or through ingested food and ultimately posed serious threat to amphibian population. In
addition, the major pollutants are absorbed through the digestive system, transported to the liver for detoxification and bio-transformation (Fasulo et al., 2015; Samantha et al., 2015; Guerriero et al., 2017, 2018; Piscopo et al., 2018). Moreover, bioaccumulation and persistence of these compounds in trophic endpoints aggravate the biological threat more adversely, as witnessed by acute and chronic poisoning of amphibians and other aquatic organisms (Pandey et al., 2005; Lajmanovich et al., 2011). Chronic sublethal effects are also clearly developed when concentrations are below the acute thresholds (Ruiz de Arcaute et al., 2018). Amphibians have adaptive plastic ability to alter their biological traits to these environmental cues for survival under new altered environment (Attademo et al., 2014; Johnson et al., 2015). In vivo inhibition or biomarkers induction, in this regard, could be considered as prominent tool to evaluate the adverse effects of these chemicals on
∗
Corresponding author. E-mail address: [email protected] (A.R. Ghosh). 1 Authors contributing equally. https://doi.org/10.1016/j.ecoenv.2020.110209 Received 20 August 2019; Received in revised form 29 November 2019; Accepted 12 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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100 mg L−1 Potassium dichromate (K2Cr2O7) – 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 mg L−1 Almix herbicide – 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 mg L−1 The measured arsenic concentrations were 9.97 ± 0.04, 20.45 ± 0.02, 30.1 ± 0.04, 40.15 ± 0.10, 50.03 ± 0.05, 59.87 ± 0.05, 70.12 ± 0.04, 80.12 ± 0.02, 90.11 ± 0.1 and 100.12 ± 0.06 mg L−1. The measured chromium concentrations were 29.87 ± 0.12, 60.26 ± 0.07, 90.23 ± 0.11, 119.88 ± 0.21, 150.19 ± 0.24, 179.96 ± 0.13, 210.33 ± 0.16, 239.92 ± 0.22, 270.3 ± 0.21 and 300.24 ± 0.18 mg L−1, respectively.
amphibians (Lajmanovich et al., 2010; Yadav et al., 2015; Pal et al., 2018). Acute toxicity testing is the prime step to determine the requirement of water quality for survival. Consequently, the acute and chronic sublethal toxicity of these chemicals at different biological levels have widely been summarized (Dinehart et al., 2010; Lajmanovich et al., 2011; Wei et al., 2014). In particularly, glutathione S-transferases (GST) involved in electrophilic metabolites conjugation with tripeptide glutathione to form water-soluble metabolite and can be used as biomarker for heavy metals/pesticides exposure in amphibian species (Brodeur et al., 2009; Lajmanovich et al., 2011; Ruiz de Arcaute et al., 2018; Pal et al., 2018). Alkaline phosphatase, a polyfunctional enzyme, involved in transphosphorylase and mineralization of the skeleton of aquatic animals and considered as commonly used biomarker of environmental contaminants including heavy metals/pesticides (Samanta et al., 2014; Pal et al., 2018). Furthermore, the chronic exposure and accumulation of metals/pesticides imposed the deleterious effects not only to exposed organism but also to other organisms including human beings (Pandey et al., 2005; Ruiz de Arcaute et al., 2018). However, information regarding toxicological paradigm of heavy metals (in particularly, arsenic and chromium)/pesticides on Indian skittering frog tadpole, Euphlyctis cyanophlyctis (Schneider, 1799) are scarce. Arsenic and chromium are widespread environmental contaminants throughout the world and their ubiquitous existence in the environment affect global health through inhalation, ingestion, and skin absorption (Brodeur et al., 2009). In particularly, the alluvial plain of the Ganga River is vastly contaminated by As pollution and Almix application in agricultural fields increased dramatically in India. Moreover, limited ecotoxicological studies have been conducted on Euphlyctis cyanophlyctis in Asia, where threats from heavy metals/pesticides are of primary concern due to agrochemical and/or industrial discharges. Additionally, to our knowledge, no studies have assessed the toxicity of As, Cr and Almix herbicide to skittering frog tadpole, E. cyanophlyctis. Our recent study demonstrated the fluoride-induced acute oxidative stress responses in Euphlyctis cyanophlyctis (Pal et al., 2018). Therefore, it is very essential to evaluate the detrimental effects of these chemicals for formulating the strategies to protect the amphibian species and other aquatic organisms as well. The present study was performed to evaluate the acute toxicity (LC50) of arsenic, chromium and commercial herbicide, Almix to Indian skittering frog tadpole, E. cyanophlyctis and to determine the biochemical endpoints. Skittering frog tadpole was selected as experimental species because it is widely available in pools, marshes and wetlands of South Asian countries. Additionally, being a predator for various invertebrates and prey for reptiles, birds and mammals, they play a pivotal role in trophic structure in the ecosystem (Greulich and Pflugmacher, 2004). Furthermore, the demand for frog meat (3.2 billion frogs/year) is rising dramatically throughout the world mainly for treatment of gastrointestinal, allergies, and as diet (Gratwicke et al., 2009; Oliveira et al., 2017).
2.2. Specimen collection and maintenance, and experimental design E. cyanophlyctis egg mass were cultured in uncontaminated pond of Crop Research and Seed Multiplication Farm premises following the protocol described by Mohanty-Hejmadi (1977). E. cyanophlyctis was identified and characterized by Zoological Survey of India, Kolkata. Hatchlings were cultured until stage 25. Hatchlings were then transported to Dept. of Environmental Science, The University of Burdwan (BU) in glass beaker containing pond water. Larvae were acclimatized in 40 L aquaria under 12 h–12 h light-dark photoperiod for 14 days (stage 30) according to BU Animal care and Use Committee guidelines. On every third day aquarium water was renewed with dechlorinated tap water. Commercial fish food was provided daily basis (Tokyu Spirulina Fish Food, TH). Water parameters namely pH, electrical conductivity (EC), dissolved oxygen (DO) and total hardness (TA) were monitored on every third day as per APHA (2005). Static-renewal exposure system was used to determine the acute toxicity tests (OECD, 2008). The test concentrations were prepared freshly from respective stock solutions and introduced into bioassay tanks (1 L) using syringes. Solution in each tank was mixed gently with a clean glass rod. For each chemical, one control and ten different concentrations were housed (each concentration has three replicate). Ten healthy and active tadpoles of similar sizes from acclimatized tanks were transferred to each 1 L tank already holding treated and untreated medium. Average total length (measured from snout to tail tip) of the tadpoles (stage 30) was 1.44 ± 0.07 mm and average weight was 0.24 ± 0.08 g. Mortality was recorded on every 6 h over 96 h by visual observation. Tadpoles were considered dead when body or tail movements were not observed after gentle prodding with a glass rod. After the acute toxicity test, the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC) were calculated to determine the endpoint of each chemicals. 2.3. Measurement of enzyme activities All control and treated tadpoles exhibited survival rate > 85% at each concentration at 96 h were collected and euthanized (stage 31–32) by immersion in 1% MS-222 according to guidelines described by American Society of Ichthyologists and Herpetologists (2004) and Howe et al. (2004). Whole tadpoles were homogenized on ice in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% KCl using a motor-driven Teflon homogenizer. Homogenates were centrifuged at 10000 g for 20 min at 4 °C, and the supernatant was used for protein, alkaline phosphatase and glutathione S-transferase analysis. GST activity was measured according to Habig et al. (1974). The reaction mixture contains 50 μl extract, 2.5 mL sample buffer, 0.3 mL reduced glutathione and 150 μL CDNB (1-chloro-2,4-dinitrobenzene) solution. The enzyme activity was measured at 340 nm using extinction coefficient ε340 = 9.6 mM−1 cm−1 and expressed as U mg protein−1. Alkaline phosphatase (ALP) activity was analyzed using MERCK kit (Merck, Mumbai, India) based on our previous published paper (Samanta et al., 2014). Briefly, 400 μl R1 reagent was added with 100 μl R2 and mixed well followed by incubation at 37 °C for 60 s. After that
2. Materials and methods 2.1. Experimental chemicals The chemicals used in this experiment are as follows: arsenic trioxide, As2O3 (purity 99%, Merck, Mumbai, IN), potassium dichromate, K2Cr2O7 (purity 99%, Merck, Mumbai, IN) and commercial Almix 20WP herbicide (DuPont India). The stock solutions were prepared in sufficient amount by dissolving weighed quantities of metallic salts in water (1000, 1000 and 2000 mg L−1 for arsenic (As3+), chromium (Cr6+) and Almix herbicide, respectively). We have used 10% hydroxide solution for dissolving As2O3 and followed by neutralization with 1 N sulfuric acid. The respective nominal test concentrations were prepared from stock solution and final exposure concentrations (T1 to T10) considered for acute toxicity test were as follows: Arsenic trioxide (As2O3) – 10, 20, 30, 40, 50, 60, 70, 80, 90 and 2
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10 μl sample was added to abovementioned mixture and reading was taken immediately using auto-analyzer for 4 min and expressed as IU/L. Total protein content was determined based on Lowry et al. (1951) and expressed as mg g−1 fresh weight using bovine serum albumin (BSA) as standard. 2.4. Safe level/concentration estimation The ‘application factor’ (AF) was used to calculate the chemical's safe concentration (SC) based on Pandey et al. (2005), Ezemonye and Tongo (2009), and Zhang et al. (2011). Generally, the 96 h LC50 data is used to measure safe concentration level. 2.5. Statistical analysis Levene's test was used to measure data homogeneity and normality at p < 0.05. SPSS statistical package was used to calculate the LC50 value (IBM Corp., Armonk, NY, USA). One-way ANOVA followed by Tukey's test (p < 0.05) was conducted to check the statistical significance between concentrations. Two-way ANOVA followed by Tukey's test (p < 0.05) was conducted to check the statistical significance between concentrations and exposure time. Pearson's correlation was conducted to check the relation between LC50 value and GST activity. Data are represented as mean ± standard deviation. Different letters indicate significant differences. 3. Results and discussion 3.1. As, Cr and almix acute toxicity The mortality of E. cyanophlyctis tadpoles was zero in control compartments and lowest Cr or Almix treatment (30 mg Cr L−1 and 200 mg Almix L−1) as well. The tadpole mortalities increased with increasing concentrations and exposure duration (Fig. 1a–c). Additionally, significant differences among different concentrations over exposure duration were recorded for As (F10,32 = 44.71, F10,32 = 270.74, F7,23 = 78.61 and F7,23 = 278.86 for 24, 48, 72 and 96 h, respectively at p < 0.001), Cr (F10,32 = 53.0, F10,32 = 161.84, F10,32 = 804.2 and F8,26 = 305.44 for 24, 48, 72 and 96 h, respectively at p < 0.001) and Almix treatment (F10,32 = 932.0, F10,32 = 598.67, F9,29 = 732.0 and F9,29 = 390.69 for 24, 48, 72 and 96 h, respectively at p < 0.001). Moreover, two-way ANOVA depicted that tadpole mortalities were significantly affected by exposure concentrations (F9,119 = 992.69, p < 0.001 for As; F9,119 = 910.59, p < 0.001 for Cr, and F9,119 = 2267.03, p < 0.001 for Almix; Table 1), exposure duration (F3,119 = 439.12, p < 0.001 for As; F3,119 = 805.40, p < 0.001 for Cr, and F3,119 = 157.09, p < 0.001 for Almix; Table 1) and their interactions (F27,119 = 20.64, p < 0.001 for As; F27,119 = 44.71, p < 0.001 for Cr, and F27,119 = 29.34, p < 0.001 for Almix; Table 1). The LC50 value (95% confidence limits) of As, Cr and Almix were decreased with increasing exposure durations (24, 48, 72 and 96 h) and their values were 73.58, 56.31, 43.58 and 32.58 mg L−1; 326.68, 224.31, 171.92 and 141.99 mg L−1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L−1, respectively (Table 2). Therefore, it is clear that As is the most toxic compound for tadpole followed by Cr and Almix. This means that a small quantity of As is lethal when compared with Cr and Almix. Additionally, the LC50 values under different exposure durations were significantly differed for each chemical (F3,11 = 126.61, p < 0.001 for As; F3,11 = 531.32, p < 0.001 for Cr, and F3,11 = 30.62, p < 0.001 for Almix; Table 2). The LC50 values of As recorded in E. cyanophlyctis tadpoles were higher than the values reported by Brodeur et al. (2009) in Rhinella arenarum tadpoles (56.61 and 50.04 mg L−1 at 48 and 96 h, respectively). The LC50 value changed greatly with increasing exposure time indicating that E. cyanophlyctis tadpoles are fairly unstable to As. This range is consistent with the findings of Loumbourdis et al. (1999) and
Fig. 1. Relationships (logarithmic) between mortality of E. cyanophlyctis tadpoles and nominal concentration of As (a), Cr (b) and Almix (c).
Selvi et al. (2003) who noticed increased mortality of Rana ridibunda with increasing cadmium concentrations. The toxicity study of Cr and Almix herbicide on amphibians is barely sufficient. The 96 h LC50 value of Cr (potassium dichromate) reported by Khangarot et al. (1985) was 100 mg L−1 for Rana hexaclectyle. In our study, the LC50 value of Cr in E. cyanophlyctis tadpole was 141.99 mg L−1, which was much lower than the value reported by Khangarot et al. (1985) in Rana hexaclectyle. This indicated that E. cyanophlyctis tadpoles are more tolerant/registrant to Cr compared with Rana hexaclectyle. This tolerance, i.e., readjustment to adverse environment can be achieved by mechanisms like metal speciation, decreased uptake, increased excretion and redistribution of metals to less sensitive target sites (Enuneku and Ezemonye, 2012; Srivastav et al., 2016). Almix, a broad-spectrum fourth generation herbicide, is used extensively in Indian agronomy 3
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Table 1 Effects of chemical concentration, exposure duration and their interactions on mortality of E. cyanophlyctis tadpoles. Different letters indicate significant differences at p < 0.001 (two-way ANOVA). Chemicals
Concentration
Exposure duration
Concentration x Exposure duration
As
Ia, IIa, IIIb, IVc, Vd, VIe, VIIf, VIIIg, IXgh, Xh F9,119 = 992.69; p < 0.001 Ia, IIa, IIIa, IVb, Vc, VId, VIIe, VIIIf, IXg, Xh F9,119 = 910.59; p < 0.001 Ia, IIa, IIIa, IVb, Vc, VId, VIIe, VIIIf, IXf, Xg F9,119 = 2267.03; p < 0.001
24 ha, 48 hb, 72 hc, 96 hd F3,119 = 439.12; p < 0.001 24 ha, 48 hb, 72 hc, 96 hd F3,119 = 805.40; p < 0.001 24 ha, 48 hb, 72 hc, 96 hd F3,119 = 157.09; p < 0.001
F27,119 = 20.64; p < 0.001
Cr Almix
Duration (h)
LC50 value (mg/L)
95% confidence limit (mg/L)
24 48 72 96
73.58a 56.31b 43.58c 32.58d
61.92 - 82.04 49.39–62.36 27.08–58.13 25.34–39.42
24 48 72 96
326.68a 224.31b 171.92c 141.99d
269.43–736.72 194.91–270.59 151.39–191.71 122.67–159.74
24 48 72 96
1297.85a 1148.22b 1033.62c 955.17d
1135.81 - 1416.69 953.74 - 1285.52 864.44–1176.89 797.28–1092.99
F27,119 = 29.34; p < 0.001
the exposed stress through accelerating protein synthesis for biotransformation (Kanbur et al., 2009; Glusczak et al., 2011). However, the differences between the groups were not found to be statistically significant for Cr. Moreover, this increase is more rapid and clear in case of Almix exposure. Additionally, changes in protein levels indicated metabolic disorders (Glusczak et al., 2011). GST is involved in detoxification process and phase II biotransformation, in which exogenous compounds (electrophilic) conjugated with endogenous macromolecule, tripeptide glutathione to form water-soluble metabolite (Lajmanovich et al., 2010; Margarido et al., 2013; Attademo et al., 2014). GST activity was significantly decreased compared with control tadpoles under As treatment (F6,20 = 5.72 at p < 0.01; one-way ANOVA; Fig. 2c). Maximum reduction was recorded at 60 mg L−1 As treatment i.e., 0.018 ± 0.011 U mg protein−1. Generally, depletion of GST activity indicated oxidative stress development and cytotoxic effects of pro-oxidant xenobiotics (Hazarika et al., 2003). Therefore, inhibition of GST activity in E. cyanophlyctis by As exposure indicated decreased capacity of tadpoles to metabolize As-induced stress and has potential to induce oxidative stress. Similar responses were observed in Scinax fuscovarius tadpoles exposed to fipronil and demonstrated that GST depletion is associated with oxidative stress and cytotoxicity (Margarido et al., 2013). Additionally, GST inhibition could be correlated with higher mortality of E. cyanophlyctis tadpoles with increasing As concentrations. Significant positive correlation (r = 0.779, p < 0.01) between LC50 value and GST activity reflecting the direct involvement of GST on tadpole mortality. Moreover, depletion of this enzyme in As treatments could indicate that GST is not involved in phase II biotransformation of As. This might be due to involvement of other phase II enzymes or because of larval stage (Ferrari et al., 2009; Margarido et al., 2013). On the contrary, the lack of significant changes in GST activities for Cr (F5,17 = 2.32 at p = 0.11; one-way ANOVA; Fig. 2c) and Almix treatments (F6,20 = 1.55 at p = 0.23; one-way ANOVA; Fig. 2c) suggested that tadpoles might have adapted and developed the alternative strategies to counteract the reactive oxygen species (ROS) and oxidative stress (Margarido et al., 2013). However, the enhanced GST activities for Cr (except 120 mg L−1) and Almix (except 200, 1000 and 1200 mg L−1) treatments, although statistically not significant, indicated that this enzyme is involved in higher biotransformation of Cr and Almix (Fig. 2c). This could probably the reason for getting lower toxicity (higher LC50 value) of these compounds, although it showed 100% mortality at 48 h for highest concentration. Negative correlation between LC50 value and GST activity (r = −0.461 and −0.19 for Cr and Almix, respectively; p > 0.05) indicated that GST plays a vital role for lowering the mortality. The observed enzyme activities were similar to that demonstrated by Attademo et al. (2014) in Leptodactylus mystacinus exposed to insecticide Lambda-cyhalothrin (LTC). However, at higher Almix treatments (1000 and 1200 mg L−1) GST activity decreased, which indicated that Almix detoxification is less resulting toxicity development. Additionally, this indicated that tadpoles were not able to withstand such concentrations properly due to GST dysfunction and resulted into direct disorder or cell damage (Greulich and Pflugmacher, 2004; Lajmanovich et al., 2011). Therefore, present findings indicated that glutathione system of E. cyanophlyctis tadpoles responds differently to
Table 2 Median lethal concentration (LC50) value in E. cyanophlyctis. Different letters indicate significant differences at p < 0.01 (one-way ANOVA; Tukey test). Chemicals
F27,119 = 44.71; p < 0.001
As
Cr
Almix
since 2006 to control the weed species like Cyperus iria, Cyperus defformis, Frimbristylis sp., Eclipta alba, Ludwigia parviflora, Cyanotis axillaris, Monochoria vaginalis, Marsilea quadrifoliata, etc. (Samanta et al., 2016), but its toxicity study to amphibians is scanty and little explored. The 96 h LC50 value of Almix herbicide was too high (955.17 mg L−1) as compared to tested metalloid and heavy metal, As and Cr (32.58 and 141.99 mg L−1, respectively). This indicated that Almix is less toxic than As and Cr to E. cyanophlyctis. However, according to the Environmental Protection Agency acute toxicity guidelines (low toxic, LC50 > 10.0 mg L−1; moderate toxic, 1 < LC50 ≤ 10 mg L-1; high toxic, LC50 < 1 mg L−1 as active ingredient), all the chemicals would be considered as lower toxic compound (US Environmental Protection Agency, 2009; Zhang et al., 2010). Similar higher 48 h LC50 value (> 333 mg L−1) have been recorded for Roundup Biactive® to four frog larvae (Litoria moorei, Lymnodynastes dorsalis, Heleioporus eyrei, Crinia insignifera) (Mann and Bidwell, 1999). Additionally, apart from this no information and study are available on acute toxicity of As, Cr and Almix on E. cyanophlyctis tadpoles till today. 3.2. Effects on enzyme activities Alkaline phosphatase is considered as biomarker and plays an important role in metabolites transport across membranes (Dubey et al., 2013; Samanta et al., 2014). Generally, ALP activities of As and Almix exposure group was increased (except T6) compared with control groups (Fig. 2a). Generally, an increase of ALP activity is a sensitive biomarker of even small cellular damage (Dubey et al., 2013), therefore elevated ALP levels under As and Almix exposure indicated stress development. On the contrary, decreased ALP levels under Cr exposure suggests that E. cyanophlyctis experienced with metabolic disorder (Kanbur et al., 2009). Therefore, present findings suggest that all chemicals have potential to cause metabolic and/or tissue damage with varying degrees. Accordingly, E. cyanophlyctis exposed to toxicants showed an increase in total protein contents compared with control (Fig. 2b). The responses could be an adaptive mechanism to compensate 4
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Fig. 2. Alkaline phosphates, total protein and glutathione S-transferase levels in E. cyanophlyctis tadpoles exposed to As (a), Cr (b) and Almix (c). Data are expressed as mean ± standard deviation. Different letters indicate significant differences at p < 0.05 (one-way ANOVA; Tukey test).
Hg2+ > Ag+ > Cu2+ > Cd2+ > Fe3+ > Al3+ > Pb2+ > Zn2+ > Ni2+ > As3+ > Cr6+ > Sn4+. This finding clearly indicated that E. cyanophlyctis tadpoles have different toxicity levels. Additionally, a large variation was recorded between different methods for each chemical. Similar large variations in safe levels for malathion and mercuric chloride in Channa punctatus has been reported by Pandey et al. (2005).
different chemicals and able to prove its sensitivity as biochemical indicator against metalloid, heavy metal and herbicide contamination. Finally, further study is required to determine the varied GST responses of E. cyanophlyctis corroborating its mechanistic pathways. 3.3. Estimation of as, Cr and almix safe concentrations
4. Conclusion
The safe concentrations (SC) estimated based on 96 h LC50 value by different methods are presented in Table 3. According to Table 3, the Almix herbicide is highly safe followed by Cr and As. The observed mortality rates were also uniform and consistent with SC values with As being more toxic than Cr and Almix. Apart from this, toxicity testing statistical endpoints (LOEC - Lowest Observed Effect Concentration and NOEC - No Observed Effect Concentration) are (presented in Fig. 3) supporting the calculated SC values. The LOEC and NOEC values were consistent with observed SC and LC50 values for As, Cr and Almix. The present findings correspond with previous observations by Luoma and Rainbow (2008), Brodeur et al. (2009), Shuhaimi-Othman et al. (2012), and Wei et al. (2015) who identified the toxicity level of different metallic ions on different tadpoles species as
Present investigation has been able to record first time the acute toxicity of As, Cr and Almix in Indian skittering frog tadpole, E. cyanophlyctis. Acute toxicity (LC50) data indicated that As is more toxic than Cr and Almix to E. cyanophlyctis tadpoles and caused mortality even at short exposure. The glutathione system of E. cyanophlyctis constitutes a sensitive biochemical indicator of contamination by As, Cr and Almix. The SC findings would provide guidance to find ‘safe’ field concentration. Additionally, these findings could help to explain population decline of frogs, inhabiting water bodies contaminated with heavy metals/herbicides and could be considered as early warning signal for heavy metals/herbicides toxicity. Finally, although present study is able to provide important information regarding As, Cr and 5
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Table 3 Estimation of safe level for E. cyanophlyctis at 96 h exposure time. Chemicals
LC50 value
Method
Application factor (AP)
Safe concentration
As
32.58
Cr
141.99
Almix
955.17
Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011) Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011) Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011)
0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2 0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2 0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2
3.258 0.3258 3.258 - 0.003258 1.63 9.89 14.199 1.4199 14.199 - 0.014199 7.10 31.73 95.517 9.5517 95.517 - 0.095517 47.76 269.90
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110209. References American Society of Ichthyologists and Herpetologists, 2004. Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research. Herpetological Animal Care and Use Committee of the ASIH, Washington, DC. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. (Washington, DC). Attademo, A.M., Peltzer, P.M., Lajmanovich, R.C., Cabagna-Zenklusen, M.C., Junges, C.M., Basso, A., 2014. Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina. Environ. Monit. Assess. 186 (1), 635–649. Brodeur, J.C., Asorey, C.M., Sztrum, A., Herkovits, J., 2009. Acute and subchronic toxicity of arsenite and zinc to tadpoles of Rhinella arenarum both alone and in combination. J. Toxicol. Environ. Health Part A 72 (14), 884–890. Dinehart, S.K., Smith, L.M., McMurry, S.T., Smith, P.N., Anderson, T.A., Haukos, D.A., 2010. Acute and chronic toxicity of Roundup weathermax and ignite 280 SL to larval Spea multiplicata and S. bombifrons from the southern high plains, USA. Environ. Pollut. 158 (8), 2610–2617. Dubey, N., Khan, A.M., Raina, R., 2013. Sub-acute deltamethrin and fluoride toxicity induced hepatic oxidative stress and biochemical alterations in rats. Bull. Environ. Contam. Toxicol. 91, 334–338. Enuneku, A.A., Ezemonye, L.I., 2012. Acute toxicity of cadmium and lead to adult toad Bufo maculates. Asian J. Biol. Life Sci. 1, 238–241. Ezemonye, L.I.N., Tongo, I., 2009. Lethal and sublethal effects of atrazine to amphibian larvae. Jordan J. Biol. Sci. 2 (1), 29–36. Fasulo, S., Guerriero, G., Cappello, S., Colasanti, M., Schettino, T., Leonzio, C., Mancini, G., Gornati, R., 2015. The Bsystem biology^ in the study of xenobiotic effects on marine organisms for evaluation of the environmental health status: biotechnological applications for potential recovery. Rev. Environ. Sci. Biotechnol. 14 (3), 339–345. Ferrari, A., Lascano, C.I., Anguiano, O.L., Pechen de D'Angelo, A.M., Venturino, A., 2009. Antioxidant responses to azinphos methyl and carbaryl during the embryonic development of the toad Rhinella (Bufo) arenarum Hensel. Aquat. Toxicol. 93, 37–44. Gabor, C.R., Perkins, H.R., Heitmann, A.T., Forsburg, Z.R., Aspbury, A.S., 2019. RoundupTM with corticosterone functions as an infodisruptor to antipredator response in tadpoles. Front. Ecol. Evol. 7, 114. https://doi.org/10.3389/fevo.2019. 00114. Glusczak, L., Loro, V.L., Pretto, A., Moraes, B.S., Raabe, A., Duarte, M.F., da Fonseca, M.B., de Menezes, C.C., Valladão, D.M., 2011. Acute exposure to glyphosate herbicide affects oxidative parameters in piava (Leporinus obtusidens). Arch. Environ. Contam. Toxicol. 61 (4), 624–630. Gratwicke, B., Evans, M.J., Jenkins, P.T., Kusrini, M.D., Moore, R.D., Sevin, J., Wildt, D.E., 2009. Is the international frog legs trade a potential vector for deadly amphibian pathogens? Front. Ecol. Environ. https://doi.org/10.1890/090111. Greulich, K., Pflugmacher, S., 2004. Uptake and effects on detoxication enzymes of cypermethrin in embryos and tadpoles of amphibians. Arch. Environ. Contam. Toxicol. 47, 489–495. Guerriero, G., Bassem, S.M., Khalil, W.K.B., Temraz, T.A., Ciarcia, G., Abdel-Gawad, F.K., 2018. Temperature changes and marine fish species (Epinephelus coioides and Sparus aurata): role of oxidative stress biomarkers in toxicological food studies. Emir. J. Food Agric. 30 (3), 205–211. Guerriero, G., Brundo, M.V., Labar, S., Bianchi, A.R., Samantha, T., Dea, R., Palumbo, G., Abdel-Gawad, F.K., De Maio, A., 2017. Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases. Environ. Sci. Pollut. Res. https://doi.org/10.1007/s11356-017-0395-2. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases: the first
Fig. 3. Acute toxicity testing statistical endpoints in E. cyanophlyctis tadpoles exposed to As, Cr and Almix (96 h).
Almix toxicity on E. cyanophlyctis, further studies like influence of environmental and biotic parameters, join toxicity, etc. are needed at different biological levels to explore the mechanistic pathways for ecological risk assessment in the wilds.
Declarations of interest None.
Author’s contribution Palas Samanta and Sandipan Pal equally contributed for this paper. They have equally responsible for designing, doing experiment, analysis, writing and interpretation. Aloke Kumar Mukherjee helped to conduct statistical analysis and interpretation. Apurba Ratan Ghosh helped to design the experiment, writing and checking grammars.
Acknowledgements The authors like to thank Head of the Environmental Science, the University of Burdwan, West Bengal, India for providing laboratory facilities. Authors are also indebted to DST-FIST for providing the instrumental facilities to the Department. Authors are also thankful to Zoological Survey of India, Kolkata for their kind help. We are also thankful to the respective reviewers for improving the quality of this paper. 6
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Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles
T
Palas Samantaa,1, Sandipan Palb,1, Aloke Kumar Mukherjeec, Apurba Ratan Ghoshd,∗ a
Department of Environmental Science, Sukanta Mahavidyalaya, Dhupguri, West Bengal, India Department of Environmental Science, Aghorekamini Prakashchandra Mahavidyalaya, India c P.G. Department of Conservation Biology, Durgapur Government College, India d Ecotoxicology Lab, Department of Environmental Science, The University of Burdwan, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arsenic Chromium Almix herbicide Euphlyctis cyanophlyctis Alkaline phosphatase Glutathione S-transferase LC50
Heavy metals and herbicide are gaining serious environmental concern in aquatic toxicology due to its adverse effects on aquatic organisms especially amphibians. Accordingly, present study first time evaluated the acute toxicity of two heavy metals [arsenic (As3+) and chromium (Cr6+)] and a herbicide (Almix) to Indian skittering frog tadpole, Euphlyctis cyanophlyctis. The LC50 values of As, Cr and Almix for 24, 48, 72 and 96 h were 73.58, 56.31, 43.58 and 32.58 mg L−1; 326.68, 224.31, 171.92 and 141.99 mg L−1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L−1, respectively. It also revealed the concentration- and time-dependent increased mortality rate under these toxicants. The safety concentrations (SC) of As, Cr and Almix to tadpoles were 3.26, 14.20 and 95.52 mg L−1, respectively. The findings disclosed that As is highly toxic to E. cyanophlyctis than Cr and Almix. Alkaline phosphatase (ALP) activity showed varied responses to exposed chemicals. In particularly, ALP activity reduced significantly for Cr treatment. Glutathione S-transferase (GST) activity in E. cyanophlyctis was significantly inhibited by As treatment (p < 0.05); however, GST activity was remain unchanged for Cr and Almix (p > 0.05). The As toxicity correlates positively with GST inhibition (r = 0.779, p < 0.01); contrarily, Cr and Almix revealed negative correlation with GST induction (r = −0.461 and −0.19, respectively; p > 0.05). This result indicated that GST play a crucial role for regulating the tadpole mortality and intoxication by As, Cr and Almix. Overall, our findings demonstrate the different levels of toxic sensitivity (adverse effects) under different toxicants on E. cyanophlyctis tadpoles. Finally, the present findings could be used as baseline information of toxicosis for metalloid, heavy metal and herbicide exposures in wild frog populations.
1. Introduction According to the International Union for Conservation of Nature (IUCN) 32.5% amphibian species were vulnerable, endangered, or critically endangered; 7.4% species were critically endangered and 43% species were experiencing the population decline (IUCN, 2004; Gabor et al., 2019). Such diminution of amphibian population is mainly due to destruction and loss of habitats, like ponds, lakes, wetlands and rivers (Greulich and Pflugmacher, 2004; Li et al., 2010). Additionally, chemical contamination mainly by heavy metals and pesticides from industrial and agricultural practices also threaten the amphibian life (Sai et al., 2015). Exposure to this contaminated water, the amphibians especially can absorb these contaminants directly from water via diffusion across the skin, gonads, and various tissues or through ingested food and ultimately posed serious threat to amphibian population. In
addition, the major pollutants are absorbed through the digestive system, transported to the liver for detoxification and bio-transformation (Fasulo et al., 2015; Samantha et al., 2015; Guerriero et al., 2017, 2018; Piscopo et al., 2018). Moreover, bioaccumulation and persistence of these compounds in trophic endpoints aggravate the biological threat more adversely, as witnessed by acute and chronic poisoning of amphibians and other aquatic organisms (Pandey et al., 2005; Lajmanovich et al., 2011). Chronic sublethal effects are also clearly developed when concentrations are below the acute thresholds (Ruiz de Arcaute et al., 2018). Amphibians have adaptive plastic ability to alter their biological traits to these environmental cues for survival under new altered environment (Attademo et al., 2014; Johnson et al., 2015). In vivo inhibition or biomarkers induction, in this regard, could be considered as prominent tool to evaluate the adverse effects of these chemicals on
∗
Corresponding author. E-mail address: [email protected] (A.R. Ghosh). 1 Authors contributing equally. https://doi.org/10.1016/j.ecoenv.2020.110209 Received 20 August 2019; Received in revised form 29 November 2019; Accepted 12 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.
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100 mg L−1 Potassium dichromate (K2Cr2O7) – 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 mg L−1 Almix herbicide – 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 mg L−1 The measured arsenic concentrations were 9.97 ± 0.04, 20.45 ± 0.02, 30.1 ± 0.04, 40.15 ± 0.10, 50.03 ± 0.05, 59.87 ± 0.05, 70.12 ± 0.04, 80.12 ± 0.02, 90.11 ± 0.1 and 100.12 ± 0.06 mg L−1. The measured chromium concentrations were 29.87 ± 0.12, 60.26 ± 0.07, 90.23 ± 0.11, 119.88 ± 0.21, 150.19 ± 0.24, 179.96 ± 0.13, 210.33 ± 0.16, 239.92 ± 0.22, 270.3 ± 0.21 and 300.24 ± 0.18 mg L−1, respectively.
amphibians (Lajmanovich et al., 2010; Yadav et al., 2015; Pal et al., 2018). Acute toxicity testing is the prime step to determine the requirement of water quality for survival. Consequently, the acute and chronic sublethal toxicity of these chemicals at different biological levels have widely been summarized (Dinehart et al., 2010; Lajmanovich et al., 2011; Wei et al., 2014). In particularly, glutathione S-transferases (GST) involved in electrophilic metabolites conjugation with tripeptide glutathione to form water-soluble metabolite and can be used as biomarker for heavy metals/pesticides exposure in amphibian species (Brodeur et al., 2009; Lajmanovich et al., 2011; Ruiz de Arcaute et al., 2018; Pal et al., 2018). Alkaline phosphatase, a polyfunctional enzyme, involved in transphosphorylase and mineralization of the skeleton of aquatic animals and considered as commonly used biomarker of environmental contaminants including heavy metals/pesticides (Samanta et al., 2014; Pal et al., 2018). Furthermore, the chronic exposure and accumulation of metals/pesticides imposed the deleterious effects not only to exposed organism but also to other organisms including human beings (Pandey et al., 2005; Ruiz de Arcaute et al., 2018). However, information regarding toxicological paradigm of heavy metals (in particularly, arsenic and chromium)/pesticides on Indian skittering frog tadpole, Euphlyctis cyanophlyctis (Schneider, 1799) are scarce. Arsenic and chromium are widespread environmental contaminants throughout the world and their ubiquitous existence in the environment affect global health through inhalation, ingestion, and skin absorption (Brodeur et al., 2009). In particularly, the alluvial plain of the Ganga River is vastly contaminated by As pollution and Almix application in agricultural fields increased dramatically in India. Moreover, limited ecotoxicological studies have been conducted on Euphlyctis cyanophlyctis in Asia, where threats from heavy metals/pesticides are of primary concern due to agrochemical and/or industrial discharges. Additionally, to our knowledge, no studies have assessed the toxicity of As, Cr and Almix herbicide to skittering frog tadpole, E. cyanophlyctis. Our recent study demonstrated the fluoride-induced acute oxidative stress responses in Euphlyctis cyanophlyctis (Pal et al., 2018). Therefore, it is very essential to evaluate the detrimental effects of these chemicals for formulating the strategies to protect the amphibian species and other aquatic organisms as well. The present study was performed to evaluate the acute toxicity (LC50) of arsenic, chromium and commercial herbicide, Almix to Indian skittering frog tadpole, E. cyanophlyctis and to determine the biochemical endpoints. Skittering frog tadpole was selected as experimental species because it is widely available in pools, marshes and wetlands of South Asian countries. Additionally, being a predator for various invertebrates and prey for reptiles, birds and mammals, they play a pivotal role in trophic structure in the ecosystem (Greulich and Pflugmacher, 2004). Furthermore, the demand for frog meat (3.2 billion frogs/year) is rising dramatically throughout the world mainly for treatment of gastrointestinal, allergies, and as diet (Gratwicke et al., 2009; Oliveira et al., 2017).
2.2. Specimen collection and maintenance, and experimental design E. cyanophlyctis egg mass were cultured in uncontaminated pond of Crop Research and Seed Multiplication Farm premises following the protocol described by Mohanty-Hejmadi (1977). E. cyanophlyctis was identified and characterized by Zoological Survey of India, Kolkata. Hatchlings were cultured until stage 25. Hatchlings were then transported to Dept. of Environmental Science, The University of Burdwan (BU) in glass beaker containing pond water. Larvae were acclimatized in 40 L aquaria under 12 h–12 h light-dark photoperiod for 14 days (stage 30) according to BU Animal care and Use Committee guidelines. On every third day aquarium water was renewed with dechlorinated tap water. Commercial fish food was provided daily basis (Tokyu Spirulina Fish Food, TH). Water parameters namely pH, electrical conductivity (EC), dissolved oxygen (DO) and total hardness (TA) were monitored on every third day as per APHA (2005). Static-renewal exposure system was used to determine the acute toxicity tests (OECD, 2008). The test concentrations were prepared freshly from respective stock solutions and introduced into bioassay tanks (1 L) using syringes. Solution in each tank was mixed gently with a clean glass rod. For each chemical, one control and ten different concentrations were housed (each concentration has three replicate). Ten healthy and active tadpoles of similar sizes from acclimatized tanks were transferred to each 1 L tank already holding treated and untreated medium. Average total length (measured from snout to tail tip) of the tadpoles (stage 30) was 1.44 ± 0.07 mm and average weight was 0.24 ± 0.08 g. Mortality was recorded on every 6 h over 96 h by visual observation. Tadpoles were considered dead when body or tail movements were not observed after gentle prodding with a glass rod. After the acute toxicity test, the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC) were calculated to determine the endpoint of each chemicals. 2.3. Measurement of enzyme activities All control and treated tadpoles exhibited survival rate > 85% at each concentration at 96 h were collected and euthanized (stage 31–32) by immersion in 1% MS-222 according to guidelines described by American Society of Ichthyologists and Herpetologists (2004) and Howe et al. (2004). Whole tadpoles were homogenized on ice in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% KCl using a motor-driven Teflon homogenizer. Homogenates were centrifuged at 10000 g for 20 min at 4 °C, and the supernatant was used for protein, alkaline phosphatase and glutathione S-transferase analysis. GST activity was measured according to Habig et al. (1974). The reaction mixture contains 50 μl extract, 2.5 mL sample buffer, 0.3 mL reduced glutathione and 150 μL CDNB (1-chloro-2,4-dinitrobenzene) solution. The enzyme activity was measured at 340 nm using extinction coefficient ε340 = 9.6 mM−1 cm−1 and expressed as U mg protein−1. Alkaline phosphatase (ALP) activity was analyzed using MERCK kit (Merck, Mumbai, India) based on our previous published paper (Samanta et al., 2014). Briefly, 400 μl R1 reagent was added with 100 μl R2 and mixed well followed by incubation at 37 °C for 60 s. After that
2. Materials and methods 2.1. Experimental chemicals The chemicals used in this experiment are as follows: arsenic trioxide, As2O3 (purity 99%, Merck, Mumbai, IN), potassium dichromate, K2Cr2O7 (purity 99%, Merck, Mumbai, IN) and commercial Almix 20WP herbicide (DuPont India). The stock solutions were prepared in sufficient amount by dissolving weighed quantities of metallic salts in water (1000, 1000 and 2000 mg L−1 for arsenic (As3+), chromium (Cr6+) and Almix herbicide, respectively). We have used 10% hydroxide solution for dissolving As2O3 and followed by neutralization with 1 N sulfuric acid. The respective nominal test concentrations were prepared from stock solution and final exposure concentrations (T1 to T10) considered for acute toxicity test were as follows: Arsenic trioxide (As2O3) – 10, 20, 30, 40, 50, 60, 70, 80, 90 and 2
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10 μl sample was added to abovementioned mixture and reading was taken immediately using auto-analyzer for 4 min and expressed as IU/L. Total protein content was determined based on Lowry et al. (1951) and expressed as mg g−1 fresh weight using bovine serum albumin (BSA) as standard. 2.4. Safe level/concentration estimation The ‘application factor’ (AF) was used to calculate the chemical's safe concentration (SC) based on Pandey et al. (2005), Ezemonye and Tongo (2009), and Zhang et al. (2011). Generally, the 96 h LC50 data is used to measure safe concentration level. 2.5. Statistical analysis Levene's test was used to measure data homogeneity and normality at p < 0.05. SPSS statistical package was used to calculate the LC50 value (IBM Corp., Armonk, NY, USA). One-way ANOVA followed by Tukey's test (p < 0.05) was conducted to check the statistical significance between concentrations. Two-way ANOVA followed by Tukey's test (p < 0.05) was conducted to check the statistical significance between concentrations and exposure time. Pearson's correlation was conducted to check the relation between LC50 value and GST activity. Data are represented as mean ± standard deviation. Different letters indicate significant differences. 3. Results and discussion 3.1. As, Cr and almix acute toxicity The mortality of E. cyanophlyctis tadpoles was zero in control compartments and lowest Cr or Almix treatment (30 mg Cr L−1 and 200 mg Almix L−1) as well. The tadpole mortalities increased with increasing concentrations and exposure duration (Fig. 1a–c). Additionally, significant differences among different concentrations over exposure duration were recorded for As (F10,32 = 44.71, F10,32 = 270.74, F7,23 = 78.61 and F7,23 = 278.86 for 24, 48, 72 and 96 h, respectively at p < 0.001), Cr (F10,32 = 53.0, F10,32 = 161.84, F10,32 = 804.2 and F8,26 = 305.44 for 24, 48, 72 and 96 h, respectively at p < 0.001) and Almix treatment (F10,32 = 932.0, F10,32 = 598.67, F9,29 = 732.0 and F9,29 = 390.69 for 24, 48, 72 and 96 h, respectively at p < 0.001). Moreover, two-way ANOVA depicted that tadpole mortalities were significantly affected by exposure concentrations (F9,119 = 992.69, p < 0.001 for As; F9,119 = 910.59, p < 0.001 for Cr, and F9,119 = 2267.03, p < 0.001 for Almix; Table 1), exposure duration (F3,119 = 439.12, p < 0.001 for As; F3,119 = 805.40, p < 0.001 for Cr, and F3,119 = 157.09, p < 0.001 for Almix; Table 1) and their interactions (F27,119 = 20.64, p < 0.001 for As; F27,119 = 44.71, p < 0.001 for Cr, and F27,119 = 29.34, p < 0.001 for Almix; Table 1). The LC50 value (95% confidence limits) of As, Cr and Almix were decreased with increasing exposure durations (24, 48, 72 and 96 h) and their values were 73.58, 56.31, 43.58 and 32.58 mg L−1; 326.68, 224.31, 171.92 and 141.99 mg L−1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L−1, respectively (Table 2). Therefore, it is clear that As is the most toxic compound for tadpole followed by Cr and Almix. This means that a small quantity of As is lethal when compared with Cr and Almix. Additionally, the LC50 values under different exposure durations were significantly differed for each chemical (F3,11 = 126.61, p < 0.001 for As; F3,11 = 531.32, p < 0.001 for Cr, and F3,11 = 30.62, p < 0.001 for Almix; Table 2). The LC50 values of As recorded in E. cyanophlyctis tadpoles were higher than the values reported by Brodeur et al. (2009) in Rhinella arenarum tadpoles (56.61 and 50.04 mg L−1 at 48 and 96 h, respectively). The LC50 value changed greatly with increasing exposure time indicating that E. cyanophlyctis tadpoles are fairly unstable to As. This range is consistent with the findings of Loumbourdis et al. (1999) and
Fig. 1. Relationships (logarithmic) between mortality of E. cyanophlyctis tadpoles and nominal concentration of As (a), Cr (b) and Almix (c).
Selvi et al. (2003) who noticed increased mortality of Rana ridibunda with increasing cadmium concentrations. The toxicity study of Cr and Almix herbicide on amphibians is barely sufficient. The 96 h LC50 value of Cr (potassium dichromate) reported by Khangarot et al. (1985) was 100 mg L−1 for Rana hexaclectyle. In our study, the LC50 value of Cr in E. cyanophlyctis tadpole was 141.99 mg L−1, which was much lower than the value reported by Khangarot et al. (1985) in Rana hexaclectyle. This indicated that E. cyanophlyctis tadpoles are more tolerant/registrant to Cr compared with Rana hexaclectyle. This tolerance, i.e., readjustment to adverse environment can be achieved by mechanisms like metal speciation, decreased uptake, increased excretion and redistribution of metals to less sensitive target sites (Enuneku and Ezemonye, 2012; Srivastav et al., 2016). Almix, a broad-spectrum fourth generation herbicide, is used extensively in Indian agronomy 3
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Table 1 Effects of chemical concentration, exposure duration and their interactions on mortality of E. cyanophlyctis tadpoles. Different letters indicate significant differences at p < 0.001 (two-way ANOVA). Chemicals
Concentration
Exposure duration
Concentration x Exposure duration
As
Ia, IIa, IIIb, IVc, Vd, VIe, VIIf, VIIIg, IXgh, Xh F9,119 = 992.69; p < 0.001 Ia, IIa, IIIa, IVb, Vc, VId, VIIe, VIIIf, IXg, Xh F9,119 = 910.59; p < 0.001 Ia, IIa, IIIa, IVb, Vc, VId, VIIe, VIIIf, IXf, Xg F9,119 = 2267.03; p < 0.001
24 ha, 48 hb, 72 hc, 96 hd F3,119 = 439.12; p < 0.001 24 ha, 48 hb, 72 hc, 96 hd F3,119 = 805.40; p < 0.001 24 ha, 48 hb, 72 hc, 96 hd F3,119 = 157.09; p < 0.001
F27,119 = 20.64; p < 0.001
Cr Almix
Duration (h)
LC50 value (mg/L)
95% confidence limit (mg/L)
24 48 72 96
73.58a 56.31b 43.58c 32.58d
61.92 - 82.04 49.39–62.36 27.08–58.13 25.34–39.42
24 48 72 96
326.68a 224.31b 171.92c 141.99d
269.43–736.72 194.91–270.59 151.39–191.71 122.67–159.74
24 48 72 96
1297.85a 1148.22b 1033.62c 955.17d
1135.81 - 1416.69 953.74 - 1285.52 864.44–1176.89 797.28–1092.99
F27,119 = 29.34; p < 0.001
the exposed stress through accelerating protein synthesis for biotransformation (Kanbur et al., 2009; Glusczak et al., 2011). However, the differences between the groups were not found to be statistically significant for Cr. Moreover, this increase is more rapid and clear in case of Almix exposure. Additionally, changes in protein levels indicated metabolic disorders (Glusczak et al., 2011). GST is involved in detoxification process and phase II biotransformation, in which exogenous compounds (electrophilic) conjugated with endogenous macromolecule, tripeptide glutathione to form water-soluble metabolite (Lajmanovich et al., 2010; Margarido et al., 2013; Attademo et al., 2014). GST activity was significantly decreased compared with control tadpoles under As treatment (F6,20 = 5.72 at p < 0.01; one-way ANOVA; Fig. 2c). Maximum reduction was recorded at 60 mg L−1 As treatment i.e., 0.018 ± 0.011 U mg protein−1. Generally, depletion of GST activity indicated oxidative stress development and cytotoxic effects of pro-oxidant xenobiotics (Hazarika et al., 2003). Therefore, inhibition of GST activity in E. cyanophlyctis by As exposure indicated decreased capacity of tadpoles to metabolize As-induced stress and has potential to induce oxidative stress. Similar responses were observed in Scinax fuscovarius tadpoles exposed to fipronil and demonstrated that GST depletion is associated with oxidative stress and cytotoxicity (Margarido et al., 2013). Additionally, GST inhibition could be correlated with higher mortality of E. cyanophlyctis tadpoles with increasing As concentrations. Significant positive correlation (r = 0.779, p < 0.01) between LC50 value and GST activity reflecting the direct involvement of GST on tadpole mortality. Moreover, depletion of this enzyme in As treatments could indicate that GST is not involved in phase II biotransformation of As. This might be due to involvement of other phase II enzymes or because of larval stage (Ferrari et al., 2009; Margarido et al., 2013). On the contrary, the lack of significant changes in GST activities for Cr (F5,17 = 2.32 at p = 0.11; one-way ANOVA; Fig. 2c) and Almix treatments (F6,20 = 1.55 at p = 0.23; one-way ANOVA; Fig. 2c) suggested that tadpoles might have adapted and developed the alternative strategies to counteract the reactive oxygen species (ROS) and oxidative stress (Margarido et al., 2013). However, the enhanced GST activities for Cr (except 120 mg L−1) and Almix (except 200, 1000 and 1200 mg L−1) treatments, although statistically not significant, indicated that this enzyme is involved in higher biotransformation of Cr and Almix (Fig. 2c). This could probably the reason for getting lower toxicity (higher LC50 value) of these compounds, although it showed 100% mortality at 48 h for highest concentration. Negative correlation between LC50 value and GST activity (r = −0.461 and −0.19 for Cr and Almix, respectively; p > 0.05) indicated that GST plays a vital role for lowering the mortality. The observed enzyme activities were similar to that demonstrated by Attademo et al. (2014) in Leptodactylus mystacinus exposed to insecticide Lambda-cyhalothrin (LTC). However, at higher Almix treatments (1000 and 1200 mg L−1) GST activity decreased, which indicated that Almix detoxification is less resulting toxicity development. Additionally, this indicated that tadpoles were not able to withstand such concentrations properly due to GST dysfunction and resulted into direct disorder or cell damage (Greulich and Pflugmacher, 2004; Lajmanovich et al., 2011). Therefore, present findings indicated that glutathione system of E. cyanophlyctis tadpoles responds differently to
Table 2 Median lethal concentration (LC50) value in E. cyanophlyctis. Different letters indicate significant differences at p < 0.01 (one-way ANOVA; Tukey test). Chemicals
F27,119 = 44.71; p < 0.001
As
Cr
Almix
since 2006 to control the weed species like Cyperus iria, Cyperus defformis, Frimbristylis sp., Eclipta alba, Ludwigia parviflora, Cyanotis axillaris, Monochoria vaginalis, Marsilea quadrifoliata, etc. (Samanta et al., 2016), but its toxicity study to amphibians is scanty and little explored. The 96 h LC50 value of Almix herbicide was too high (955.17 mg L−1) as compared to tested metalloid and heavy metal, As and Cr (32.58 and 141.99 mg L−1, respectively). This indicated that Almix is less toxic than As and Cr to E. cyanophlyctis. However, according to the Environmental Protection Agency acute toxicity guidelines (low toxic, LC50 > 10.0 mg L−1; moderate toxic, 1 < LC50 ≤ 10 mg L-1; high toxic, LC50 < 1 mg L−1 as active ingredient), all the chemicals would be considered as lower toxic compound (US Environmental Protection Agency, 2009; Zhang et al., 2010). Similar higher 48 h LC50 value (> 333 mg L−1) have been recorded for Roundup Biactive® to four frog larvae (Litoria moorei, Lymnodynastes dorsalis, Heleioporus eyrei, Crinia insignifera) (Mann and Bidwell, 1999). Additionally, apart from this no information and study are available on acute toxicity of As, Cr and Almix on E. cyanophlyctis tadpoles till today. 3.2. Effects on enzyme activities Alkaline phosphatase is considered as biomarker and plays an important role in metabolites transport across membranes (Dubey et al., 2013; Samanta et al., 2014). Generally, ALP activities of As and Almix exposure group was increased (except T6) compared with control groups (Fig. 2a). Generally, an increase of ALP activity is a sensitive biomarker of even small cellular damage (Dubey et al., 2013), therefore elevated ALP levels under As and Almix exposure indicated stress development. On the contrary, decreased ALP levels under Cr exposure suggests that E. cyanophlyctis experienced with metabolic disorder (Kanbur et al., 2009). Therefore, present findings suggest that all chemicals have potential to cause metabolic and/or tissue damage with varying degrees. Accordingly, E. cyanophlyctis exposed to toxicants showed an increase in total protein contents compared with control (Fig. 2b). The responses could be an adaptive mechanism to compensate 4
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Fig. 2. Alkaline phosphates, total protein and glutathione S-transferase levels in E. cyanophlyctis tadpoles exposed to As (a), Cr (b) and Almix (c). Data are expressed as mean ± standard deviation. Different letters indicate significant differences at p < 0.05 (one-way ANOVA; Tukey test).
Hg2+ > Ag+ > Cu2+ > Cd2+ > Fe3+ > Al3+ > Pb2+ > Zn2+ > Ni2+ > As3+ > Cr6+ > Sn4+. This finding clearly indicated that E. cyanophlyctis tadpoles have different toxicity levels. Additionally, a large variation was recorded between different methods for each chemical. Similar large variations in safe levels for malathion and mercuric chloride in Channa punctatus has been reported by Pandey et al. (2005).
different chemicals and able to prove its sensitivity as biochemical indicator against metalloid, heavy metal and herbicide contamination. Finally, further study is required to determine the varied GST responses of E. cyanophlyctis corroborating its mechanistic pathways. 3.3. Estimation of as, Cr and almix safe concentrations
4. Conclusion
The safe concentrations (SC) estimated based on 96 h LC50 value by different methods are presented in Table 3. According to Table 3, the Almix herbicide is highly safe followed by Cr and As. The observed mortality rates were also uniform and consistent with SC values with As being more toxic than Cr and Almix. Apart from this, toxicity testing statistical endpoints (LOEC - Lowest Observed Effect Concentration and NOEC - No Observed Effect Concentration) are (presented in Fig. 3) supporting the calculated SC values. The LOEC and NOEC values were consistent with observed SC and LC50 values for As, Cr and Almix. The present findings correspond with previous observations by Luoma and Rainbow (2008), Brodeur et al. (2009), Shuhaimi-Othman et al. (2012), and Wei et al. (2015) who identified the toxicity level of different metallic ions on different tadpoles species as
Present investigation has been able to record first time the acute toxicity of As, Cr and Almix in Indian skittering frog tadpole, E. cyanophlyctis. Acute toxicity (LC50) data indicated that As is more toxic than Cr and Almix to E. cyanophlyctis tadpoles and caused mortality even at short exposure. The glutathione system of E. cyanophlyctis constitutes a sensitive biochemical indicator of contamination by As, Cr and Almix. The SC findings would provide guidance to find ‘safe’ field concentration. Additionally, these findings could help to explain population decline of frogs, inhabiting water bodies contaminated with heavy metals/herbicides and could be considered as early warning signal for heavy metals/herbicides toxicity. Finally, although present study is able to provide important information regarding As, Cr and 5
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Table 3 Estimation of safe level for E. cyanophlyctis at 96 h exposure time. Chemicals
LC50 value
Method
Application factor (AP)
Safe concentration
As
32.58
Cr
141.99
Almix
955.17
Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011) Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011) Ezemonye and Tongo (2009) Pandey et al. (2005) Pandey et al. (2005) Pandey et al. (2005) Zhang et al. (2011)
0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2 0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2 0.1 0.01 0.1 to 0.0001 5% of 96-h LC50 (48 h LC50 * 0.3)/(24 h LC50/48 h LC50)2
3.258 0.3258 3.258 - 0.003258 1.63 9.89 14.199 1.4199 14.199 - 0.014199 7.10 31.73 95.517 9.5517 95.517 - 0.095517 47.76 269.90
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110209. References American Society of Ichthyologists and Herpetologists, 2004. Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research. Herpetological Animal Care and Use Committee of the ASIH, Washington, DC. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. (Washington, DC). Attademo, A.M., Peltzer, P.M., Lajmanovich, R.C., Cabagna-Zenklusen, M.C., Junges, C.M., Basso, A., 2014. Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina. Environ. Monit. Assess. 186 (1), 635–649. Brodeur, J.C., Asorey, C.M., Sztrum, A., Herkovits, J., 2009. Acute and subchronic toxicity of arsenite and zinc to tadpoles of Rhinella arenarum both alone and in combination. J. Toxicol. Environ. Health Part A 72 (14), 884–890. Dinehart, S.K., Smith, L.M., McMurry, S.T., Smith, P.N., Anderson, T.A., Haukos, D.A., 2010. Acute and chronic toxicity of Roundup weathermax and ignite 280 SL to larval Spea multiplicata and S. bombifrons from the southern high plains, USA. Environ. Pollut. 158 (8), 2610–2617. Dubey, N., Khan, A.M., Raina, R., 2013. Sub-acute deltamethrin and fluoride toxicity induced hepatic oxidative stress and biochemical alterations in rats. Bull. Environ. Contam. Toxicol. 91, 334–338. Enuneku, A.A., Ezemonye, L.I., 2012. Acute toxicity of cadmium and lead to adult toad Bufo maculates. Asian J. Biol. Life Sci. 1, 238–241. Ezemonye, L.I.N., Tongo, I., 2009. Lethal and sublethal effects of atrazine to amphibian larvae. Jordan J. Biol. Sci. 2 (1), 29–36. Fasulo, S., Guerriero, G., Cappello, S., Colasanti, M., Schettino, T., Leonzio, C., Mancini, G., Gornati, R., 2015. The Bsystem biology^ in the study of xenobiotic effects on marine organisms for evaluation of the environmental health status: biotechnological applications for potential recovery. Rev. Environ. Sci. Biotechnol. 14 (3), 339–345. Ferrari, A., Lascano, C.I., Anguiano, O.L., Pechen de D'Angelo, A.M., Venturino, A., 2009. Antioxidant responses to azinphos methyl and carbaryl during the embryonic development of the toad Rhinella (Bufo) arenarum Hensel. Aquat. Toxicol. 93, 37–44. Gabor, C.R., Perkins, H.R., Heitmann, A.T., Forsburg, Z.R., Aspbury, A.S., 2019. RoundupTM with corticosterone functions as an infodisruptor to antipredator response in tadpoles. Front. Ecol. Evol. 7, 114. https://doi.org/10.3389/fevo.2019. 00114. Glusczak, L., Loro, V.L., Pretto, A., Moraes, B.S., Raabe, A., Duarte, M.F., da Fonseca, M.B., de Menezes, C.C., Valladão, D.M., 2011. Acute exposure to glyphosate herbicide affects oxidative parameters in piava (Leporinus obtusidens). Arch. Environ. Contam. Toxicol. 61 (4), 624–630. Gratwicke, B., Evans, M.J., Jenkins, P.T., Kusrini, M.D., Moore, R.D., Sevin, J., Wildt, D.E., 2009. Is the international frog legs trade a potential vector for deadly amphibian pathogens? Front. Ecol. Environ. https://doi.org/10.1890/090111. Greulich, K., Pflugmacher, S., 2004. Uptake and effects on detoxication enzymes of cypermethrin in embryos and tadpoles of amphibians. Arch. Environ. Contam. Toxicol. 47, 489–495. Guerriero, G., Bassem, S.M., Khalil, W.K.B., Temraz, T.A., Ciarcia, G., Abdel-Gawad, F.K., 2018. Temperature changes and marine fish species (Epinephelus coioides and Sparus aurata): role of oxidative stress biomarkers in toxicological food studies. Emir. J. Food Agric. 30 (3), 205–211. Guerriero, G., Brundo, M.V., Labar, S., Bianchi, A.R., Samantha, T., Dea, R., Palumbo, G., Abdel-Gawad, F.K., De Maio, A., 2017. Frog (Pelophylax bergeri, Günther 1986) endocrine disruption assessment: characterization and role of skin poly(ADP-ribose) polymerases. Environ. Sci. Pollut. Res. https://doi.org/10.1007/s11356-017-0395-2. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases: the first
Fig. 3. Acute toxicity testing statistical endpoints in E. cyanophlyctis tadpoles exposed to As, Cr and Almix (96 h).
Almix toxicity on E. cyanophlyctis, further studies like influence of environmental and biotic parameters, join toxicity, etc. are needed at different biological levels to explore the mechanistic pathways for ecological risk assessment in the wilds.
Declarations of interest None.
Author’s contribution Palas Samanta and Sandipan Pal equally contributed for this paper. They have equally responsible for designing, doing experiment, analysis, writing and interpretation. Aloke Kumar Mukherjee helped to conduct statistical analysis and interpretation. Apurba Ratan Ghosh helped to design the experiment, writing and checking grammars.
Acknowledgements The authors like to thank Head of the Environmental Science, the University of Burdwan, West Bengal, India for providing laboratory facilities. Authors are also indebted to DST-FIST for providing the instrumental facilities to the Department. Authors are also thankful to Zoological Survey of India, Kolkata for their kind help. We are also thankful to the respective reviewers for improving the quality of this paper. 6
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