Environmentally relevant concentrations of butachlor inhibited the development of the green toad (Bufotes sitibundus) during the incubation period

doi:10.21203/rs.3.rs-5277838/v1

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Environmentally relevant concentrations of butachlor inhibited the development of the green toad (Bufotes sitibundus) during the incubation period

https://doi.org/10.21203/rs.3.rs-5277838/v1

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Butachlor is one of the most widely used herbicides in agricultural areas throughout the world. Studies have measured the toxicity of butachlor in single life stages of amphibians, however, less attention has been paid to the impacts of this herbicide on various life stages. Therefore, we collected the eggs of the green toad Bufotes sitibundus from a clean environment with no history of pesticides. We then exposed the collected eggs to the environmentally relevant concentrations of butachlor and measured the growth, survival, and development of green toad during the incubation period. We also measured five different responses i.e., length at the beginning of metamorphosis (LBM), length at the formation of forelimb bud (LFF), length at the end of metamorphosis (LEM), weight at the beginning of metamorphosis (WBM), and weight at the end of metamorphosis (WEM) during the incubation period. The survival rate showed an indirect relationship with butachlor concentrations. The longest development duration was recorded for toads reared in the control (110 days), while the shortest duration (9 days) was observed in the highest butachlor concentration (i.e., 1.5 mg/L), with the highest mortality recorded in this treatment. with all the eggs being died at days 11 and 9 after the start of incubation. We found a significant difference between the survival of toads in butachlor treatments and the control group (P < 0.05). We observed a significant difference between treatments and the control group in LBM, LFF, LEM, and WEM (P < 0.05), except WBM (P > 0.05). Our findings highlight that butachlor at environmentally relevant concentrations inhibited the development of the green toad and mitigated the survival of eggs and larvae, resulting in the unsuccessful completion of the development before metamorphosis as a critical life stage.

Endocrine-disrupting herbicide

Amphibian species

Development stage

Metamorphosis

Reproductive success

Herbicide and pesticide applications have caused drastic declines in amphibian populations existing in agricultural fields (Hayes et al. 2010). These chemicals are linked to a wide range of biological and ecological issues (Rick et al. 2005), including biodiversity (Khan & Law 2005), abundance (Wanger et al. 2023), and reproduction (Thompson et al. 2023) across amphibian species. Agrochemicals such as herbicides impact amphibians in their different life stages e.g., eggs (Trudeau et al. 2020), larvae (Camacho-Rozo & Camacho-Reyes 2022), metaphors (Agostini et al. 2020), and adults (Moutinho et al. 2020). These chemicals can induce endocrine-disrupting effects and cause significant changes in reproduction, resulting in lowering fecundity and reducing the number of individuals in populations (Babalola 2016). Given that amphibians have permeable skin, chemicals such as herbicides can easily pass through their skin and impact critical pathways such as hormone-regulated developmental stages during larvae and metamorphosis (Norris 2024). As paddy fields and rice farms provide a good source of food and habitat for amphibians, various pesticides used in these locations pose serious risks to amphibians (Kittusamy et al. 2014). Thus, the study of these effects in different life stages of amphibians provides significant insights into the short- and long-term consequences of widely used pesticides in the biology and ecology of these species.

While there is a wide range of pesticides used in paddy fields and rice farms, the application of herbicides such as butachlor has gained attention in these agricultural areas in recent years (Lukanov et al. 2024). Butachlor is being largely used in rice farms to control trash and unwanted plants competing with target crops i.e., rice (Rao et al. 2012). The residual of this herbicide harms various plant and animal species existing in the water and soil of the farms (Janaki et al. 2016). Studies have shown that butachlor residual half-life differs approximately between 3 and 30 days in different soil environments (Pal et al. 2006). Farmers use butachlor in paddy fields after transplanting the rice in the prepared farms to avoid growing weeds that compete with rice for light and nutrients. It is evident that butachlor causes neurotoxic and genotoxic effects in organisms living in paddy farms such as snails, toads, frogs, and fish (Liu et al. 2011). Long-term exposure of snakehead fish (Channa punctata) to butachlor was found to accumulate in internal organs (Tilak et al. 2007). Butachlor exposure reduced the growth and survival of tadpoles in frogs and increased the development stage of the larvae before metamorphosis (Li et al. 2016). However, less attention has been paid to measuring the potential negative effects of butachlor in toads at environmentally realistic concentrations.

Exposure to butachlor has been shown to have detrimental effects on various amphibian species, significantly impacting their growth, survival, and development. Studies have demonstrated that this herbicide can cause developmental delays, reduced growth rates, and increased mortality among amphibians. For example, research on the Luzon wart frog (Fejervarya vittigera) found that butachlor exposure at environmentally relevant concentrations could induce feminization in male frogs, leading to disruptions in reproductive development and potential population declines (Salvani & Jumawan 2023). Similarly, Indian bullfrog (Hoplobatrachus tigerinus) larvae exposed to butachlor exhibited significant reductions in growth and survival rates, highlighting the herbicide's capacity to severely impair early life stages (Sharma et al. 2016). The herbicide's prevalence in agricultural landscapes poses a substantial risk, particularly to amphibians that rely on waterlogged environments such as paddy fields for breeding and development. Notably, toads, which often inhabit these rice farms, are particularly vulnerable during their larval stages when they are in constant contact with contaminated water. While most studies have measured the toxicity of pesticides on amphibians in a single life stage e.g., only eggs or only tadpoles, less attention has been paid to examining the potential impacts of these toxicants on various developmental stages during the incubation period.

The green toad (Bufotes sitibundus) is a widely distributed amphibian species that inhabits a variety of environments, from arid regions to agricultural landscapes like paddy fields. Known for its adaptability, the green toad breeds in temporary water bodies, laying eggs that develop into tadpoles before metamorphosing into adult toads (Mazgajska & Mazgajski 2020). This species plays a crucial role in controlling insect populations and maintaining ecological balance within its habitats. However, the green toad is increasingly vulnerable to environmental pollutants, particularly pesticides used in agriculture. Studies have shown that exposure to various pesticides can significantly affect the development, growth, and survival of B. sitibundus. For instance, pesticides can cause developmental abnormalities, reduce growth rates, and increase mortality in tadpoles, leading to population declines. In Iran, where B. sitibundus is widely distributed, the extensive use of butachlor in agricultural practices raises concerns about its potential impact on this species. The application of butachlor in rice farms across various regions of Iran coincides with the breeding season of the green toad, increasing the likelihood of exposure during critical developmental periods. This could lead to localized population declines and disrupt the ecological roles that these toads play in their environments. Given the ecological importance of B. sitibundus and its wide distribution in Iran, understanding the impacts of butachlor and other agricultural chemicals on this species is crucial for conservation efforts. Therefore, in this study, we aimed to measure the growth, survival, and developmental stages of B. sitibundus in response to environmentally relevant concentrations of butachlor. We hypothesized that environmentally relevant concentrations of butachlor decrease the survival rate and growth of B. sitibundus in different life stages i.e., eggs, larvae, and metamorphosed individuals. We also hypothesized that five different measurements during the incubation period i.e., length at the beginning of metamorphosis (LBM), length at the formation of forelimb bud (LFF), length at the end of metamorphosis (LEM), weight at the beginning of metamorphosis (WBM), and weight at the end of metamorphosis (WEM) of B. sitibundus decrease with the increase of butachlor concentrations.

Study animal

B. sitibundus is widely distributed across a broad range extending from Greece eastwards through Turkey, Syria, Jordan, and Lebanon, and further into Iraq and Iran. This species spans the Caucasus and Russia, reaching Kazakhstan, with isolated populations reported in Denmark, northern Germany, and southern Sweden (Fakharzadeh & Hosseinzadeh 2021). Historically classified as Bufotes viridis with three subspecies, recent molecular studies have redefined the taxon, recognizing B. sitibundus as a distinct species. In Iran, B. sitibundus is primarily found in the western and central regions, previously identified under B. viridis with subspecies distributed in different provinces. This species is a member of the B. viridis complex, which is notable as the only known anuran group comprising diploid, tetraploid, and triploid taxa that reproduce bisexually and are widespread throughout the Palearctic. In Iran, all three ploidy levels (2n, 3n, 4n) have been documented, although cytogenetic evidence from several populations indicates that B. sitibundus in this region is predominantly diploid (Fakharzadeh et al. 2015).

Sampling

Kazzaz (49^◦26'15''E and 34^◦00'10''N) is a village in Pol-e Doab Rural District, Zalian District, Shazand County, Markazi Province, Iran. At the 2006 census, its population was 2,148, in 536 families. In April 2022, we collected B. sitibundus egg clutches (3500 eggs from the same clutches) from one of the B. sitibundus breeding habitats (under the permission of the Ethics Committee of Arak University (IR.ARAKU.REC.1401.080) that were free of any butachlor herbicide contamination. To avoid any influence from pesticides, the eggs were obtained from a spring with clean water in an area where there are no agricultural lands or evidence for herbicides in this area. We collected the eggs right after the deposition and immediately transferred them to the lab.

Herbicide concentrations

We based the butachlor concentrations on the environmentally relevant concentrations previously reported in the environment. Kaur et al. (2017) reported the range of butachlor concentrations from 0.121 ± 0.051 µg/L to 0.357 ± 0.338 µg/L in the water of rice farms after herbicide spraying. Concentrations of butachlor reported to be lethal to 50% of amphibian embryos and larvae (LC50s) range from 0.53 mg/L (96 h, Microhyla ornata) to 2.67 mg/L (24 h, Polypedates megacephalus) (Abigail et al. 2014; Li et al. 2016). We therefore used a series of butachlor concentrations of 1.5, 1, 0.8, 0.6, 0.4, and 0.1 mg/L, plus a control group with no added herbicide.

To prepare the desired concentrations of butachlor for the experiment, we first obtained emulsion butachlor with a purity of 65% (CAS No. 23184-66-9) from (Behsam Alborz). A stock solution of butachlor was prepared by dissolving the required amount of the herbicide in deionized water to achieve a stock concentration. The stock solution was then serially diluted using deionized water to obtain the working concentrations needed for the experiment. We stored the stock solution in a dark, airtight glass container at 4°C throughout the incubation period to prevent degradation and maintain stability. Fresh working solutions were prepared weekly by diluting the stock solution immediately before use to ensure the accuracy and consistency of the herbicide concentrations in the experimental setups. All glassware and containers used in the preparation and storage of butachlor solutions were thoroughly cleaned and rinsed with deionized water to avoid contamination.

Experimental design

The green toad’s eggs were housed in seven polyethylene tubes, each containing 500 eggs and approximately 5L of water. Six of the tubes contained different concentrations of butachlor: 1.5 mg/L, 1 mg/L, 0.8 mg/L, 0.6 mg/L, 0.4 mg/L, and 0.1 mg/L, while the seventh tube, serving as the control group, contained clean habitat water. We started the incubation period by adding the prepared concentrations to each tube. The water in each tube was renewed every seven days during the experiment, and a new stock of herbicide concentrations was added to each treatment. To ensure consistent oxygen levels across all treatments during the incubation period, oxygen was continuously supplied to the rearing tubes using an air pump. Since the rearing area was in an open space with natural light and subject to the ambient light and dark cycles, we did not interfere with these conditions. Additionally, the temperature and humidity of the environment, as well as the temperature of the rearing containers, were recorded throughout the entire incubation period (see Fig. S1-S3). To maximize the similarity of the study environment to the natural breeding habitat, the study was conducted in an open environment influenced by natural temperature and light-dark cycles. Temperature and humidity fluctuations during the day and night were recorded throughout the entire incubation period.

The incubation period continued until the completion of the metamorphosis of all individuals. Given that the development of the oral structure of larvae is completed at Gosner stage 25 (Gosner 1960), cooked lettuce leaves, boiled potatoes, and cooked spinach were used to feed the larvae at this stage (two times/day).

Developmental stage parameters

We measured the growth, death rates, length at the beginning of metamorphosis (LBM (mm)), length at the formation of forelimb bud (LFF (mm)), length at the end of metamorphosis (LEM (mm)), weight at the beginning of metamorphosis (WBM (gr)), weight at the end of metamorphosis (WEM (gr)) for a sample size of three in each phase (Gosner, 1960).

Parameters such as the length of the incubation period, the number of metamorphoses, the number of eggs that perished before entering the embryonic stage, the number of embryos that perished before entering the larval stage, and the number of larvae that did not complete metamorphosis were compared in different groups during the incubation period.

Data analyses

We compared the length of the incubation period, the number of metamorphoses, the number of dead eggs before entering the embryonic stage, the number of dead embryos before entering the larval stage, and the number of larvae that did not complete metamorphosis across different treatments and the control group. Survival analyses were conducted using the Kaplan-Meier (1958) method, a non-parametric statistic used to estimate the survival function from lifetime data. This method is particularly useful for handling censored data, such as when a subject leaves the study before an event occurs. The Kaplan-Meier survival curves, which display survival probabilities over time as a series of declining horizontal steps, were compared across different concentrations using the Mantel-Haenszel log-rank test (Harrington & Fleming 1982). This test compares the survival distributions of two or more groups by testing the null hypothesis that there is no difference in survival experiences across the groups.

We also used Multivariate Analysis of Variance (MANOVA) and Univariate Analysis of Variance (ANOVA) to compare the length and weight of metamorphosed larvae (LBM, LFF, LEM, WBM, WEM) across different treatment groups. MANOVA was employed to assess multiple dependent variables simultaneously, particularly useful when these variables are correlated. Eta squared was calculated as an effect size measure to quantify the proportion of variance in the dependent variables attributable to the independent variable(s), with values around 0.01 and 0.06 indicating small to medium effects, and values greater than 0.14 indicating large effects. Post-hoc comparisons were made using the Tukey HSD (Honestly Significant Difference) test to determine which specific group means differed significantly from each other in the various butachlor treatments and the control.

Incubation period of B. sitibundus influenced by butachlor

We found the longest incubation period at 110 days in the control group with individuals completing their different life stages during the whole incubation period (Table 1). At the two highest butachlor concentrations i.e., 1 and 1.5 mg/L, the toad’s eggs experienced the hatching stage but all died a few days after at days 11 and 9, respectively. We found an indirect relationship between the butachlor concentration and the time that individuals survived. As the concentration increased from 0.1 to 1.5 mg/L, the duration that individuals continued development stages decreased (i.e., from 76 days in 0.1 mg/L to 9 days in 1.5 mg/L).

Table 1

Incubation period of *B. sitibundus* across different butachlor treatments
Group	Incubation period (days)
BTC 1.5	9
BTC 1	11
BTC 0.8	72
BTC 0.6	67
BTC 0.4	70
BTC 0.1	76
Control	110

Development stage of B. sitibundus in different butachlor concentrations

We observed that B. sitibundus eggs experienced the hatching stage across all treatments (plus signs in Fig. 1). Relative to the control group, the time at which toads started metamorphosis delayed at concentrations of 0.1, 0.4, 0.6, and 0.8 mg/L, and no eggs reached the metamorphosis stage in the two highest concentrations of butachlor i.e., 1 and 1.5 mg/L (Fig. 1). Similarly, relative to the control group, the time at which the first metamorphosed toad was observed delayed at 0.1, 0.4, 0.6, and 0.8 mg/L concentrations, and no toads were able to reach this stage in 1 and 1.5 mg/L butachlor (Fig. 1).

Survival rate of B. sitibundus during the incubation period

We observed the highest survival rate of toads in the control group during the incubation period (Fig. 2). The survival rate showed a direct relationship with the butachlor treatments, and with the increase in concentrations, the survival rate decreased. Toads showed a sharp drop in survival at the two highest butachlor concentrations compared to the other treatments (Fig. 2). The differences in survival rates were further confirmed by the multiple and pairwise Mantel-Haenszel log-rank test (see Table S1-S2).

Toxicity of butachlor to B. sitibundus in different measurements during the incubation period

All five measurements i.e., the length at the beginning of metamorphosis (LBM), the length at the formation of forelimb bud (LFF), length at the end of metamorphosis (LEM), weight at the beginning of metamorphosis (WBM), weight at the end of metamorphosis (WEM) showed the highest values in the control group. The results of MANOVA and Eta squared show significant differences in five measurements between groups (see Table S3 and Table S4). We observed a gradual reduction in butachlor treatments, and with the increase in concentrations, these measurements decreased (Fig. 3). Using ANOVA and Tukey HSD, we found significant differences between butachlor treatments and the control group in all measurements, except for the WBM (see Table S5-S9)

We found strong evidence that environmentally relevant concentrations of butachlor impact different life stages of B. sitibundus during the incubation period. Our findings highlight that concentrations of butachlor reduced the survival and growth of individuals during the incubation period. We also found that relative to the control and other treatments, butachlor concentrations higher than 1 mg/L inhibited the development of the toad’s eggs a few days after the hatching. These results are in agreement with our hypothesis that various concentrations of butachlor decrease the capability of green toad to complete its development in different life stages during the incubation period. We also observed that five different measurements of B. sitibundus i.e., LBM, LFF, LEM, WBM, and WEM decrease across all butachlor treatments relative to the control group. This is in line with our hypothesis that elevated concentrations of butachlor decrease these responses during the incubation period. Our findings show that environmentally relevant concentrations of butachlor delay different development stages of B. sitibundus relative to the control group. In summary, we found that butachlor impacts various life stages of green toads from eggs to metamorphosis, and this impact increases with the elevation of concentrations.

Investigation of surface water near agricultural soils in Hainan, China showed that butachlor is one of the pollutants that often exceed their regulatory threshold, about 0.0001 mg/L (Tan et al. 2020). Therefore, these pesticides can persist for a long time in nature and will impact a wide range of organisms’ life stages. These adverse effects are more severe in the case of amphibians who are precariously dependent on the aquatic environment and spend their entire embryonic and larval stages in the aquatic environment (Shuman-Goodier et al. 2021). Shuman-Goodier et al. (2021) showed that exposure to 0.002, 0.02, and 0.2 mg/L of butachlor led to a decrease in transcripts related to metabolic processes, anatomical structure development, immune system function, and stress response in the cane toad (Rhinella marina). The result of our current study showed that the concentration of 1.5 mg/L of butachlor, which is the highest concentration used in the study design, inhibited the development and the hatching of the eggs. In the two highest concentrations i.e., 1 and 1.5 mg/L, butachlor led to a high mortality, and all the individuals died at days 9 to 11 of the incubation period.

Butachlor is widely used in rice farms and paddy fields where many amphibians spend their breeding time and find their mate (Liu et al. 2011). As the application of this herbicide is at the same time as the breeding in spring, many non-target species including toads are influenced by butachlor. This suggests that almost all toads or frogs that make reproductive calls in the rice paddies before spraying butachlor will not reproduce successfully (Ok et al. 2012). Literature has reported that low concentrations of herbicides have a wide range of disorders in the growth of eggs, hatching, and development of tailless amphibians (Ok et al. 2012). Similarly, we found that the survival and growth of individuals exposed to butachlor are more sensitive than the other responses measured. From the results obtained in this study, what seems to be more important is the survival and growth indicators in the treated groups. The survival index from the day of lying eggs to the time for metamorphosis decreased relative to the control groups in all studied treatments. In this way, with the increase in the concentration of butachlor, survival decreased significantly. In line with our findings, Semlitsch et al. (2000) found that pesticides impact the most sensitive life stages of amphibians, including larval and metamorphosis stages, and affect the reproductive performance of adults over subsequent generations. Thus, even at the lowest concentrations, butachlor delays critical life stages such as metamorphosis which later impacts the whole population.

Studies have found that tolerant amphibians can overcome chemical pollution and cope with polluted environments to some extent (Semlitsch et al. 2000). However, at high concentrations these species may not be able to detoxify and reduce the toxicity effects, and therefore, experience low survival rate or even death. Studies reported that there is a direct correlation between the size of adult amphibians and their size during the larval time (Earl & Semlitsch 2013) and larval size before metamorphosis (Cabrera-Guzman et al. 2013), and with the increase of embryonic phase duration, the size of the larvae increases, resulting in enhancing the tolerance of individuals later in juvenile and adult stages (Luquet et al. 2011; Luquet et al. 2013). Shuman-Goodier et al. (2021) showed that cane toads (Rhinella marina) exposed to butachlor grew slower and weighed less than the control group, and the length of the incubation period was also reduced, which is consistent with the results obtained in this study.

There are many mechanisms underlying the toxicity of butachlor to amphibians. However, determining the exact mechanism influencing the growth and survival varies depending on the species, life stage, and type of toxicant. Butachlor decreases the size of thyroid cells by affecting the thyroid gland and thus affects the growth and weight gain of amphibian larvae (Shuman-Goodier et al. 2021). Thyroid gland dysfunction was observed in X. laevis frogs exposed to a concentration of 0.1 mg/L butachlor (Li et al. 2016). Fejervarya limnocharis showed slower growth in exposure to butachlor at concentrations of 0.025 and 0.1 mg/L relative to the control (Liu et al., 2011). Thus, the existence of such a chemical compound in nature, even if it does not endanger the survival of some larvae, will greatly reduce their competence in adulthood. Our findings are consistent with Shuman-Goodier et al. (2021) who found strong evidence that exposure to environmentally relevant concentrations of butachlor disrupts the development and growth of sugarcane toads, especially in the early stages of development. Further research is needed to determine the exact mechanisms by which butachlor impacts the growth and survival rate of the green toad. Also, future research should measure the molecular and cellular pathways at which butachlor causes damage to DNA and critical cells and organs.

Our study provides a strong line of evidence showing that butachlor at environmentally relevant concentrations decreases the growth and survival of green toads in different life stages. We found an indirect relationship between the concentrations of butachlor and the survival of eggs, larvae, and metamorphosed individuals. Our findings confirm that exposure to butachlor has irreparable effects on green toads, which can be divided into two primary and final steps. First, if amphibians are exposed to high concentrations of this herbicide in nature, they will not reproduce successfully because the eggs will either die before hatching or their metamorphosis could be inhibited. Second, if they succeed in the metamorphosis by being exposed to low concentrations of butachlor, they will have much less competence in adulthood due to the high stresses they have endured and will most likely perish or at least fail to reproduce upon re-exposure to toxins. Such conditions will be much more problematic in areas that are facing water stress and the habitats are limited to agricultural areas, and may greatly reduce or cause the extinction of amphibian populations in those areas. If our results occur in nature, butachlor could reduce the number of survived eggs and hatching rate, leading to lowering the number of individuals in the population. Moreover, butachlor especially at rice farms and paddy fields delays green toad’s metamorphosis which is costly for this species. More research regarding the molecular and cellular effects of butachlor are suggested to better find the mechanisms and pathways by which green toads are affected in the environment.

Author contribution

A. Pesarakloo: Conceptualization, Writing original-first draft, and Experimental design, Z. Zarehi, and SJ Mirkamali: Material preparation, data collection, and analysis, M. Esmaeilbeigi: Revision and Graphical analyses, All: Revision, Editing, and Approval for the final manuscript.

Funding

Notapplicable.

Data availability

All data generated or analyzed during this study are included in this published article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval

Under the permission of the Ethics Committee of Arak University (IR.ARAKU.REC.1401.080)

Consent to participate

Not applicable.

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Competing interests

The authors declare no competing interests.

Abigail MEA, Samuel MS, Chidambaram R (2014) Hexavalent chromium biosorption studies using Penicillium griseofulvum MSR1 a novel isolate from tannery effluent site: Box–Behnken optimization, equilibrium, kinetics and thermodynamic studies. J Taiwan Ins Chem Eng 49:156–164
Agostini MG, Roesler I, Bonetto C, Ronco AE, Bilenca D (2020) Pesticides in the real world: The consequences of GMO-based intensive agriculture on native amphibians. Biol Conserv 241: 108355. https://doi.org/10.1016/j.biocon.2019.108355
Atar N, Olgun A, Wang S, Liu S (2011) Adsorption on anionic dyes on boron industry waste in single and binary solutions using batch and fixed bed systems. J Chem Eng Data 56: 508–516
Babalola OO (2016) Ecotoxicological and potential endocrine effects of selected aquatic herbicides on life stages of the African clawed frog, Xenopus laevis, Stellenbosch University
Beebee TJC, Griffiths RA (2005) The amphibian decline crisis: a watershed for conservation biology? Biol Conserv 125: 271–285.
Bijlsma R, Loeschcke V (2012) Genetic erosion impedes adaptive responses to stressful environments. Evol Appl 5: 117e129.
Blaustein AR, Betsy A (2007) Amphibian Population Declines: Evolutionary Considerations. BioScience 57(5): 437–444. https://doi.org/10.1641/B570517
Blaustein AR, Han BA, Relyea RA, Johnson PT, Buck JC, Gervasi SS, Kats LB (2011) The complexity of amphibian population declines: understanding the role of cofactors in driving amphibian losses. Ann N Y Acad Sci 1223: 108–119. https://doi.org/10.1111/j.1749-6632.2010.05909.x.
Cabrera-Guzman E, Crossland MR, Brown GP, Shine R (2013) Larger body size at metamorphosis enhances survival, growth and performance of young cane toads (Rhinella marina). PloS One 8. https://doi.org/10.1371/journal.pone.0070121.
Camacho-Rozo CP, Camacho-Reyes JA (2022) Effect of agricultural pesticides and land use intensification on amphibian larval development. In IntechOpen https://doi.org/10.5772/intechopen.106268
Chen CS, Wu TW, Wang HL, Wu SH, Tien CJ (2014) The ability of immobilized bacterial consortia and strains from river biofilms to degrade the carbamate pesticide methomyl. Int J Environ Sci Technol. doi:10.1007/s13762-014-0675-z
Coutellec MA, Barata C (2011) An introduction to evolutionary processes in ecotoxicology. Ecotoxicology 20: 493e496.
Cox C, Surgan M (2006) Unidentified inert ingredients in pesticides: Implications for human and environmental health. Environ Health Perspect 114:1803–1806.
Dill GM, Sammons RD, Feng PCC, Kohn F, Kretzmer K, Mehrsheikh A, Bleeke M, Honegger, JL, Farmer D, Wright D, Haupfear EA (2010) Glyphosate: Discovery, development, applications, and properties. In Nandula VK, ed, Glyphosate Resistance in Crops and Weeds. John Wiley & Sons, Hoboken, NJ, USA, pp 1–33.
Earl J, Semlitsch R (2013) Carryover effects in amphibians: are characteristics of the larval habitat needed to predict juvenile survival? Ecol Appl 23 (6): 1429e1442. http://www.esajournals.org/doi/abs/10.1890/12-1235.1.
European Commission (2002) Review report for the active substance glyphosate. Health & Consumer Protection Directorate, Brussels, Belgium.
Fakharzadeh F, Darvish J, Kami HG, Ghassemzadeh F, Rastegar-Pouyani E, Stöck M (2015) Discovery of triploidy in Palearctic green toads (Anura: Bufonidae) from Iran with indications for a reproductive system involving diploids and triploids. Zool Anz 255: 25 – 31.http://dx.doi.org/10.1016/j.jcz.2015.01.001
Fakharzadeh F, Hosseinzadeh MS (2021) Overview of taxonomy and prediction potential distribution of Bufotes sitibundus (Anura: Bufonidae) using environmental factors. J Wild Biodivers 5(3): 21–34. https://doi.org/10.22120/jwb.2021.138255.119
Fedorenkova A, Vonk J (2012) Ranking ecological risks of multiple chemical stressors on amphibians. Environ Toxicol Chem 31: 1416e1421.
Fenoll J, Ruiz E, Flores P, Hellin P, Navarro S (2011) Reduction of the movement and persistence of pesticides in soil through common agronomic practices. Chemosphere 85:1375–1382
Green DM (2003) The ecology of extinction: population fluctuation and decline in amphibians. Biol Conserv 111: 331e343.
Harrington DP, Fleming TR (1982) A class of rank test procedures for censored survival data. Biometrika 69: 553–566.
Hayes TB, Falso P, Gallipeau S, Stice M (2010) The cause of global amphibian declines: A developmental endocrinologist's perspective. J Experiment Biol 213(6): 921–933. https://doi.org/10.1242/jeb.040865
Heimlich R, Fernandez-Cornejo J, McBride W, Klotz-Ingram C, Jans S, Brooks N (2000) Genetically engineered crops: Has adoption reduced pesticide use? Agr Outlook 273:13–17.
Janaki P, Chinnusamy C, Meena S, Shanmugasundaram R, Sakthivel N (2016) Effect of repeated and long term application of butachlor on its dissipation kinetics in rice soil. Asian JChem 28(10): 2277–2282. http://dx.doi.org/10.14233/ajchem.2016.19967
Kaplan EL, Meier P (1958) Nonparametric estimation from incomplete observations. J Am Stat Assoc 53(282): 457–481.
Kaur P, Randhawa SK, Duhan A, Bhullar MS (2017) Influence of Long Term Application of Butachlor on its Dissipation and Harvest Residues in Soil and Rice. Bull Environ Contam Toxicol 98: 874e880. https://doi.org/10.1007/s00128-017-2070-1.
Khan MZ, Law FCP (2005) Adverse effects of pesticides and related chemicals on enzyme and hormone systems of fish, amphibians, and reptiles: A review. Proc Pakistan Acad Sci 42(4): 315–323.
Kittusamy G, Kandaswamy C, Kandan N, Kumar S (2014) Pesticide residues in two frog species in a paddy agroecosystem in Palakkad District, Kerala, India. Bull Environ Contam Toxicol 93(6): 728–734. https://doi.org/10.1007/s00128-014-1351-1
Li S, Li M, Wang Q, Gui W, Zhu G (2016) Exposure to butachlor causes thyroid endocrine disruption and promotion of metamorphosis in Xenopus laevis .Chemosphere 152: 158e165. https://doi.org/10.1016/j.chemosphere.2016.02.098.
Liu WY, Wang CY, Wang TS, Fellers GM, Lai BC, Kam YC (2011) Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan. Ecotoxicology 20(2): 377–384. https://doi.org/10.1007/s10646-010-0589-6
Liu WY, Wang CY, Wang TS, Fellers GM, Lai BC, Kam YC (2011) Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan. Ecotoxicology 20: 377e384. https://doi.org/10.1007/s10646-010-0589-6.
Lukanov S, Kolev A, Dimitrova B, Popgeorgiev G (2024) Rice fields as important habitats for three anuran species—Significance and implications for conservation. Animals 14(1): 106. https://doi.org/10.3390/ani14010106
Luquet E et al. (2011) Consequences of genetic erosion on fitness and phenotypic plasticity in European tree frog populations (Hyla arborea). J. Evol. Biol 24: 99e110.
Luquet E et al. (2013) Within- and among-population impact of genetic erosion on adult fitness-related traits in the European tree frog Hyla arborea. Heredity (Edinb) 110: 347e354.
Mazgajska J, Mazgajski TD (2020) Two amphibian species in the urban environment: changes in the occurrence, spawning phenology, and adult condition of common and green toads. Urban Ecosyst 23(2):170–179. https://doi.org/10.1080/24750263.2020.1744743
Mirbagheri SA, Monfared SA (2009) Pesticide transport and transformation modelling in soil column and groundwater contamination prediction. Int J Environ Sci Tech 6(2): 233–242
Moutinho MF, de Almeida EA, Espíndola E.LG, de Oliveira AG (2020) Herbicides employed in sugarcane plantations have lethal and sublethal effects on larval Boana pardalis (Amphibia, Hylidae). Ecotoxicology 29(7): 1043–1051. https://doi.org/10.1007/s10646-020-02226-z
Norris DO (2024) Environmental influences on hormones and reproduction in amphibians. In Hormones and Reproduction of Vertebrates (2nd ed., Vol. 2, pp. 257–289). Elsevier. https://doi.org/10.1016/B978-0-443-16020-2.00003-6
OECD (Organisation for Economic Co-operation and Development., 2006. OECD environmental indicators. OECD Publishing, Paris.
Ok J, Doan NH, Watanabe H, Thuyet DQ, Boulange J (2012) Behavior of butachlor and pyrazosulfuron-ethyl in paddy water using micro paddy lysimeters under different temperature conditions in spring and summer. Bull. Environ. Contam. Toxicol 89: 306e311. https://doi.org/10.1007/s00128-012-0700-1.
Pal R, Das P, Chakrabarti K, Chakraborty A, Chowdhury A (2006) Butachlor degradation in tropical soils: Effect of application rate, biotic-abiotic interactions, and soil conditions. J Environ Sci Health B 41(7): 1103–1113. https://doi.org/10.1080/03601230600851141
Pesarakloo A, Hoseinabad M (2023) Food composition of a breeding population of the green toad, Bufotes sitibundus (Pallas, 1771) (Anura: Bufonidae), from Iran. Biol Bull Russ Academy of Sciences 50: 157–161. https://doi.org/10.1134/S1062359022700029
Rao PC, Lakshmi CSR, Madhavi M, Swapna G, Sireesha A (2012) Butachlor dissipation in rice grown soil and its residues in grain. J Agric Food Chem 44(2): 84–87. https://doi.org/10.1007/s00128-014-1351-1
Relyea RA, Schoeppner NM, Hoverman J T (2005) Pesticides and amphibians: The importance of community context. Ecol Appli 15(4): 1125–1134. https://doi.org/10.1890/04-0559
Ribeiro R, Lopes I (2013) Contaminant driven genetic erosion and associated hypotheses on alleles loss, reduced population growth rate and increased susceptibility to future stressors: an essay. Ecotoxicology 22: 889e899.
Samuel MS, Abigail MEA, Ramalingam C (2015a) Isotherm modelling, kinetic study and optimization of batch parameters using response surface methodology for effective removal of Cr(VI) using fungal biomass. PLoS ONE 10(3): 0116884
Samuel MS, Abigail MEA, Ramalingam C (2015b) Biosorption of Cr(VI) by Ceratocystis paradoxa MSR2 using isotherm modelling, kinetic study and optimization of batch parameters using response surface methodology. PLoS ONE 10(3): 0118999
Santhanam N, Samuel MS, Ramalingam C (2014) Electronic waste an emerging threat to the environment of urban India. J Environ Health Sci Eng 12: 36
Sarma PJ, Kumar R, Pakshirajan K (2015) Batach and continuos removal of copper and lead from aqueous solution using cheaply available agricultural waste materials. Int J Environ Res 9(2): 635–648
Schmeller D, Schregel J, Veith M (2007) The importance of heterozygosity in a frog's life. Naturwissenschaften 94: 360–366. http://dx.doi.org/10.1007/s00114-006-0205-z.
Semlitsch RD, Bridges CM, Welch AM (2000) Genetic variation and a fitness tradeoff in the tolerance of gray treefrog (Hyla versicolor) tadpoles to the insecticide carbaryl. Oecologia 125:179–185.
Semlitsch RD, Bridges CM, Welch AM (2000) Genetic variation and a fitness tradeoff in the tolerance of gray treefrog (Hyla versicolor) tadpoles to the insecticide carbaryl. Oecologia 125: 179e185.
Semlitsch RD, Earl J (2013) Carryover effects in amphibians: Are characteristics of the larval habitat needed to predict juvenile survival? Ecol App 23(6):1429–42
Shuman-Goodier ME, Singleton GR, Forsman AM, Hines S, Christodoulides N, Daniels KD, Propper CR (2021) Developmental assays using invasive cane toads, Rhinella marina, reveal safety concerns of a common formulation of the rice herbicide,butachlor. Environ Pollut 272: 115955 https://doi.org/10.1016/j.envpol.2020.115955
Smith MA, Green DM (2005) Dispersal and the metapopulation paradigm in amphibian ecology and conservation: are all amphibian populations metapopulations? Ecography. (Cop.) 28: 110e128.
Tajik R, Alimoradian A, Jamalian M, Shamsi M, Moradzadeh R, Ansari Asl B, Asafari M (2021) Lead and cadmium contaminations in fruits and vegetables, and arsenic in rice: A cross-sectional study on risk assessment in Iran. Iran J Toxicol 15(2): 73–82. https://doi.org/10.32598/IJT.15.2.784.1
Tan H, Li Q, Zhang H, Wu C, Zhao S, Deng X, Li Y (2020) Pesticide residues in agricultural topsoil from the Hainan tropical riverside basin: determination, distribution, and relationships with planting patterns and surface water. Sci Total Environ 722: 137856. https://doi.org/10.1016/j.scitotenv.2020.13785.
Thompson CM, Sweeney MR, Popescu VD (2022) Carryover effects of pesticide exposure and pond drying on performance, behavior, and sex ratios in a pool breeding amphibian. J Zool 318(1): 56–65. https://doi.org/10.1111/jzo.12975
Tilak KS, Veeraiah K, Bhaskara Thathaji P, Butchiram MS (2007) Toxicity studies of butachlor to the freshwater fish Channa punctata (Bloch). J Environ Biol 28(2 Suppl): 485–487. PMID: 17929770
Trudeau VL, Thomson P, Zhang WS, Reynaud S, Navarro-Martin L, Langlois VS (2020) Agrochemicals disrupt multiple endocrine axes in amphibians. Molecular and Cellular Endocrinology 513: 110861. https://doi.org/10.1016/j.mce.2020.110861
Wanger TC, Brook BW, Evans T, Tscharntke T (2023) Pesticides reduce tropical amphibian and reptile diversity in agricultural landscapes in Indonesia. Peer J 11: e15046. https://doi.org/10.7717/peerj.15046
Zeisset I, Beebee TJC (2008) Amphibian phylogeography: a model for understanding historical aspects of species distributions. Heredity (Edinb): 101: 109e119.

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Environmentally relevant concentrations of butachlor inhibited the development of the green toad (Bufotes sitibundus) during the incubation period

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