Toxicity and Genotoxicity of Imidacloprid in Tadpoles of Leptodactylus Latrans and Physalaemus Cuvieri (Anura: Leptodactylidae)


 Imidacloprid is a neonicotinoid insecticide widely used worldwide, but which can cause adverse effects on non-target organisms, especially in aquatic environments. This study aimed to evaluate the chronic toxicity of an insecticide-based imidacloprid in amphibians, using Leptodactylus latrans and Physalaemus cuvieri tadpoles. The parameters of survival, swimming activity, body size, damage to body structures and genotoxicity for both species were analyzed; and the ecological risk of this insecticide calculated. Chronic short-term assay was carried out for 168 h (7 days) and five concentrations of imidacloprid, between 3 and 300 µg L-1, were tested. The insecticide did not affect the tadpoles survival tadpoles; however, both species tested showed smaller body size, damage to the mouth and intestine and the induction of micronuclei and other erythrocytes nuclear abnormalities after exposure to imidacloprid-based herbicide. Insecticide exposure affected the swimming activity in L. latrans, which contributes to the greater sensitivity of L. latrans to imidacloprid when compared to P. cuvieri. All parameters analyzed indicated that the insecticide presents an ecological risk for both species at concentrations greater than 3 µg L-1. This demonstrates the genotoxicity of the insecticide imadacloprid, which can contribute to the population decline of L. latrans and P. cuvieri species in natural systems.


Introduction
The neonicotinoid insecticides were launched in the 90s and almost immediately became a preference over organophosphates and carbamates in the control of herbivorous insects. Currently neonicotinoids are the most used class of insecticides worldwide (Simon-Delso et al. 2015; Bakker et al. 2020;Borsuah et al. 2020). The widespread use of neonicotinoids has become a global environmental issue since the destination, behavior and effects of its residues are poorly understood and scarce (Pietrzak et al. 2020). In addition, overuse often occurs, without adding bene t to cultivation. (Simon-Delso et al. 2015).
The most widely used neonicotinoid worldwide is imidacloprid (Jeschke et al. 2011;Pietrzak et al. 2019;IBAMA 2021). This insecticide is considered moderately toxic, and is indicated for foliar application in crops such as lettuce, coffee, sugar cane, beans, tobacco, corn, tomatoes, wheat and grapes (ANVISA 2021). In insects, it has a neurotoxic action, chemically interacting to mimic the action of acetylcholine, binding to the nicotinic receptors (nAChRs) of this important neurotransmitter (Kagabu 2011). By acting selectively on insect nAChRs (Liu and Casida 1993;Tomizawa and Casida 2005), this interaction triggers excessive neuron stimulation resulting in the death of these animals (Simon-Delso et al. 2015).
Imidacloprid has low sorption and slow soil degradation, and great potential for leaching into groundwater (Hashimoto et al. 2020;Pietrzak et al. 2020). Although imidacloprid is not suitable for the water to be consumed, it can reach water bodies through spraying, draining or leaching (Wiggins et al. 2018; Chen et al. 2019). This insecticide is persistent in water, with a half-life of 30 days and may not be readily biodegradable (Hladik et al. 2018). Due to these characteristics, it is a pesticide often found in surface water ( Some countries have standardized limits for imidacloprid in water. In Canada, the maximum allowed concentration of imidacloprid for the protection of aquatic life is 0.23 µg L − 1 ; in the USA, the limit established by the Environmental Protection Agency (EPA) is 1.05 µg L − 1 ; and in Netherlands the environmental risk index is 0.2 µg L − 1 to acute toxicity and 0.067 µg L − 1 to chronic toxicity (Hrynyk et al. 2018). The use of imidacloprid is prohibited in the eld in the European Union, and its use is allowed only in greenhouses, since 2018 (Jactel et al. 2019). In Brazil, there is a limit of 300 µg L − 1 in drinking water in Rio Grande do Sul state (Brazil 2014), but this is the only restriction de ned in the country.
The high solubility of neonicotinoids in water can cause adverse effects in non-target organisms, such as in vertebrates, such as genotoxicity, cytotoxicity, changes in immune functions, reduced growth or even reproductive failure (Gibbons et al. 2015;Hrynyk et al. 2018). The toxicity of imidacloprid has been veri ed for several non-target organisms belonging to aquatic communities, such as aquatic insects (Kobashi et  Amphibians, among vertebrates, are considered excellent bioindicators, as they have permeable skin and are sensitive to changes in environmental conditions (Haddad et al. 2008). In addition, amphibians are considered of special interest because they are in decline in population and with the increase in endangered species (Beasley 2019), which has recently been associated with pesticides exposure (Agostini et al. 2020). These organisms become more susceptible to pollutants because they occupy a transitional niche between terrestrial and aquatic ecosystems (Mason et al. 2013;Jing et al. 2017), mainly during the reproductive stage, which occurs mainly in a humid environment; which coincides with the periods of application of pesticides for large crops, during spring and summer (Tavalieri et al. 2020).
Toxicological studies using native species are important to assess the sensitivity of these species and to understand the impacts of toxic substances. Leptodactylus latrans (Fitzinger 1826) and Physalaemus cuvieri (Steffen 1815) are two species native to South America, with reproductive strategy in foam nests (Mijares et al. 2010;Sá et al. 2014). Even listed as least concern (LC) by the International Union for Conservation of Nature Red List of Threatened Species (IUCN 2021), both species, L. latrans and P. cuvieri, showed sensitivity to glyphosate, such as development, behavioral and morphological changes, genotoxic effects and lethality (Bach et al. 2018;Herek et al. 2020); and glyphosate combined with 2,4-D was also toxic to L. latrans (Pavan et al. 2021).
Leptodactylus latrans, popularly known as butter frog, has a wide geographical distribution in South America, east of the Andes. It is very adaptable, occurring in different types of habitats, both in preserved areas and in disturbed and modi ed environments. Their spawns are deposited in large foam nests produced on the surface of the water (Heyer et al. 2010). Physalaemus cuvieri popularly known as dog-frog, inhabit open and anthropized areas (Kwet and Di Bernardo 1999;Eterovick and Sazima 2004). The organisms are widely distributed in Brazil, Argentina, and Paraguay, preferably reproducing in temporary bodies of water; they reproduce preferentially in temporary bodies of water, where they deposit their spawning also in foam nests, close to the vegetation that borders the lagoons, on the water surface (Mijares et al. 2010).
The aim of this study was to determine the chronic toxicity of environmentally relevant concentrations of imidacloprid-based insecticide in tadpoles of L. latrans and P. cuvieri by assessing survival, swimming activity, body size, damage to body structures and genotoxicity. We still calculate the ecological risk to understand the effects of this insecticide on amphibians.

Experimental design and experimental conditions
Ten tadpoles in development stage 25 were transferred to 500 ml glass containers, and each container was considered an experimental unit. The assays were conducted in triplicate, totaling 30 tadpoles per treatment. The tadpoles used in the tests had complete mouth formation, swimming capacity, and similar and normal length and mass. The tadpoles of L. latrans had a length of 13.25 ± 0.36 mm and mass body of 0.035 ± 0.008 g, and P. cuvieri had, in average, 16.60 mm ± 0.60 mm and 0.070 g ± 0.011 g.
Chronic short-term exposure was carried out, with a total duration of 168 hours (7 days) according to ASTM STP 1443 (Herkovits and Perez-Coll 2003), as a static test and the tadpoles were fed daily as previously described. Tadpoles were exposed to ve water treatments with a

Survival, swimming activity and body size and structures
Tadpole survival was checked every 24 h, when live and dead tadpoles were recorded. Dead tadpoles were removed from the containers. Swimming activity was also recorded every 24 h by qualitative observation. The tadpoles were gently stimulated with a glass rod and the observed behavior was noted. The behavior was classi ed as: a) swimming activity equal to the control, b) lethargy (reduced swimming activity in relation to the control), c) hyperactivity (increased swimming activity in relation to the control), d) unresponsive (without the occurrence of movements) and e) spasms (tremors and convulsions).
At the end of the assay period, the tadpoles were euthanized with lidocaine (5%) following the rules of the National Council for Animal Control and Experimentation (CONCEA 2015). Body size measurements of 10 tadpoles from each treatment were measured. The total length (mm) was veri ed using a digital caliper (150 mm MTX®, Moscow, Russia) from the face to the tail, and the mass (g) using a precision scale (AUX320, Shimadzu Analítica®, Kyoto, Japan). In addition, body structures were evaluated. Changes in the mouth (denticles or morphology) and changes in the intestine (edema or morphology) were determined. Digital images of morphological traits were taken using a digital camera (P510®, Nikon, Tokyo, Japan) and damage in body structures were determined using a stereomicroscope (SZ51®, Olympus, Tokyo, Japan).

Micronucleus Assay and Other Nuclear Erythrocytic Abnormalities
For genotoxic analysis, a drop of blood was obtained from 10 tadpoles from each insecticide and control treatments.

Ecological risk analysis
The chronic risk analysis was based on the Hazard Quotient (HQ) method for aquatic animals de ned by the United States Environmental Agency (USEPA 2020). The calculation is made by the equation: EEC/NOEC, where EEC is the estimated environmental concentration and NOEC represents no observed effect concentration. We used EEC as the limit allowed for imidacloprid in the state of Rio Grande do Sul, Brazil, which is 300 µg L − 1 . The result of the equation was compared with the level of concern (LOC) of the United States Environmental Protection Agency (USEPA). The LOC indicates whether a pesticide has a potential risk of causing adverse effects to non-target organisms (USEPA 2021). The LOC reference value for chronic risk for aquatic animals is 1; therefore, values greater than 1 represent the existence of adverse effects of the contaminant. We also determined the lowest observed effect concentration (LOEC), which represents the lowest concentration able to cause toxic effects. Thus, it was possible to infer the maximum acceptable toxicant concentration (MATC), which corresponds to the average of the LOEC and NOEC values. When it was not possible to statistically calculate the NOEC, the LOEC values were used to determine HQ, and we de ned that the MATC was equal to LOEC.

Statistical analyses
The data obtained were previously analyzed for normality by the Kolmogorov-Smirnov test (K-S) and for homogeneity of variances by the Barlett test. With the assumptions accepted, the analysis of variance (ANOVA) was performed, and the treatment means were compared with the control treatment by the Dunnet test (p < 0.05). Statistical analyses and graphs were performed using Statistic 8.0 and GraphPad Prism 7.0 software, respectively. In the ANOVA results we use acronyms to identify the tested species, Ll for Leptodactylus latrans and Pc for P. cuvieri.

Results
Exposure to imidacloprid-based insecticide did not signi cantly in uence the survival of L. latrans and P. cuvieri after 168h in chronic assay. In L. latrans the survival of the exposed tadpoles was on average 84.67% (F Ll5,12 = 1.16; p = 0.380) and in P. cuvieri was 100% in all treatments. The data are shown as supplementary material (Online Resource 1).
Swimming activity Only L. latrans showed changes in swimming activity when exposed to imidacloprid (Online Resource 3). The most frequent behavior was lethargy (30.67% of the exposed tadpoles), followed by hyperactivity (20.67%), and spasms (18.67%). Unresponsive was observed in 18% of treated tadpoles (Fig 4).
Except for the karyolysis (KA) in P. cuvieri, all other ANEs as nuclear bubble/bud (NB), binucleated cell (BC), notched nucleus (NN), and lobed nucleus (LN) were found both in L.atrans and P. cuvieri, mainly at the concentration of 300 µg L -1 ( Table 1). The most frequent ANEs were notched nucleus and lobed nucleus, which were signi cantly higher from the concentration of 30 µg L -1 of imidacloprid (Table 1).

Ecological risk analysis
All parameters analyzed presented ecological risk for L. latrans and P. cuvieri (Table 2), considering hazard quotiente (HQ) values higher than the reference value LOC (level of concern) = 1, as determined by USEPA (2021). The maximum acceptable toxicant concentration (MATC) was between 3.00 and 9.49 μg L -1 of imidacloprid for L. latrans, and between 3.00 e 9.49 μg L -1 for P. cuvieri (Table 2).  Changes in the development of tadpoles were veri ed by the shorter length and body mass observed in both species, with L. latrans being more sensitive to imidacloprid than P. cuvieri. Low development has already been related to imidacloprid, in concentrations well below those associated with mortality (Gibbons et al. 2015).
In stressful situations, such as in the presence of contaminants, the expenditure of resources to try to tolerate the presence of pesticides can reduce the resources available for growth (DiGiacopo and Hua 2020). The impairment of the development of tadpoles can also contribute to greater predation in natural environments, both due to the smaller size (e.g. Carlson  The structural integrity of the intestine guarantees functional tness for digestion, a fundamental process for nutrient absorption (Sun et al. 2018). This process requires the presence of the intestinal microbiota, which is related to the stress response and can be affected even by pesticides . Previous studies have shown that imidacloprid is capable of inducing microbiota dysbiosis in crabs (Hong et al. 2020) and mice ). According to Yang et al (2020), the reduction of the intestinal barrier weakens the organ to toxic substances. Thus, over time, the tadpoles would have di culties in feeding.
In natural populations of amphibians, morphologically malformed individuals generally constitute a small fraction of less than 2% (Ouellet 2000); however, the high number of damage (> 50% of individuals) by imidacloprid observed in this study demonstrates the high toxicity of Despite changes in the morphology of both tadpoles, only L. latrans showed changes in swimming activity when exposed to imidacloprid. In addition to dietary changes due to damage to the mouth and intestine, energy expenditure in hyperactivity or even less food intake due to lethargy behavior may have contributed to the lower growth and greater sensitivity by L. latrans to imidacloprid. The change in activity swimming by exposure to imidacloprid has also been found in other amphibians (eg, Lee- sensitive in Boana pulchella tadpoles exposed to pirimicarb-based formulation , as observed in the present study for both species. Any external factor that affects cell proliferation, differentiation or apoptosis can produce embryotoxic or teratogenic effects, and can result in permanent congenital malformations, functional abnormalities or even the death of individuals (Gilbert 2006).
Based on the morphological traits, swimming activity and genotoxicity, the ecological risk analysis indicated that the maximum acceptable concentration of imidacloprid for L. latrans and P. cuvieri is 3 μg L -1 . Above this concentration, both species may show damage to body structures, and L. latrans, smaller body size and changes in swimming activity. Above 9.49 μg L -1 P. cuvieri has smaller body size, in addition to genotoxic cell damage in both species. The fact that chronic short-term assays is long enough to cause cytotoxic damage to cells in tadpoles shows how toxic pesticides used in environmentally relevant concentrations and allowed in Brazil are toxic to amphibians. Considering the response of non-target species that live in aquatic environments, such as amphibians, the allowed concentration for imidacloprid in water should be 3 μg L -1 , which is 100 times less than the concentration allowed in the state of Rio Grande do Sul, Brazil. 300 μg L -1 (Brazil 2014). This demonstrates the need for further studies focusing on limit concentrations at different trophic levels to subsidize legislation and protect aquatic life.
Although L. latrans and P. cuvieri belong to the same family (Leptodactylidae), and have a wide distribution in South America and adaptability to different habitats (Heyer et al. 2010;Mijares et al. 2010), L. latrans tadpoles showed sensitivity greater when exposed to imidacloprid, and this is the rst study that characterizes this differentiation. This is important to determine the choice of environmental bioindicator species that can be, at the same time, easily found, and are sensitive to contaminants.
In the tadpole phase, amphibians are unable to escape exposure to environments contaminated with neocotinoids, as they use these habitats for larval development until they reach the appropriate stage to survive in the terrestrial environment (Robinson et al. 2017).
Neonicotinoids such as imidacloprid have high persistence and toxicity, and impact on trophic levels in aquatic environments, as they are able to decrease the biomass of organisms, harming the dynamics of the food chain, especially of higher-level consumers (Yamamuro et al. 2019). For this reason, conservation measures regulated in legislation for national applicability are needed, which currently does not exist in the country where the study was carried out. Still, showing the toxicity of this insecticide for anuran amphibians and the existence of other neonicotinoids, further studies are needed to detail the risks of these pesticides in populations of anurans.

Conclusion
We found that environmentally relevant concentrations of the neonicotinoid imidacloprid induced changes in the development of L. latrans and P. cuvieri. Morphological and genotoxic changes were observed in both species, however L. latrans was more sensitive to the insecticide than P. cuvieri. Imidacloprid presents a high ecological risk for the two species studied, where the maximum acceptable concentration of this insecticide is 3 μg L -1 , 100 times below what is allowed by law in Brazil. Thus, we emphasize the importance of conservation actions associated with the review or creation of speci c legislation that correlate the impacts of pesticides on the extinction of anuran amphibians. Total length (mm, ▲ ) and body mass (g, • ) of Leptodactylus latrans (a) and Physalaemus cuvieri (b) tadpoles in the control (0) and exposed for 168 h to different concentrations of herbicide-based imidacloprid Percentage of occurrence of damage in the mouth, intestine and total damage (mouth + intestine) in tadpoles of Leptodactylus latrans (a, c, e) and Physalaemus cuvieri (b, d, f) in the control and exposed to different concentrations of imidacloprid based-herbicide for 168 h. Bars represent mean ± SE. Different letters indicate signi cant differences by the Tukey test (p < 0.05) Frequency of lethargy (a), hyperactivity (b), spasm (c) and unresponsive (d), during the swimming activity of Leptodactylus latrans tadpoles exposed to imidacloprid-based herbicide for 168 h. Bars represent mean ± SE. Different letters indicate signi cant differences by the Tukey test (p < 0.05).

Supplementary Files
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