Toxicity and genotoxicity of imidacloprid in the tadpoles of Leptodactylus luctator and Physalaemus cuvieri (Anura: Leptodactylidae)

Imidacloprid is a neonicotinoid insecticide used to control agricultural pests around the world. This pesticide can have adverse effects on non-target organisms, especially in aquatic environments. The present study evaluated the toxicity of an imidacloprid-based insecticide in amphibians, using Leptodactylus luctator and Physalaemus cuvieri tadpoles as study models. Spawning of both species were collected within less than 24 h of oviposition from a non-agricultural land at Erechim, Rio Grande do Sul state, Brazil. Survival, swimming activity, body size, morphological malformations, and genotoxic parameters were analyzed at laboratory conditions. A short-term assay was conducted over 168 h (7 days) with five different concentrations of imidacloprid (3–300 µg L−1) being tested. The insecticide did not affect survival, although the tadpoles of both species presented reduced body size, malformed oral and intestine structures, and micronuclei and other erythrocyte nuclear abnormalities following exposure to this imidacloprid-based compound. Exposure also affected swimming activity in L. luctator, which reflected the greater sensitivity of L. luctator to imidacloprid in comparison with P. cuvieri. The swimming activity, body size, and malformations observed in L. luctator and the morphological malformations found in P. cuvieri indicated that even the lowest tested concentration of the insecticide were harmful to amphibians. At concentrations of over 3 μg L−1, P. cuvieri presents a smaller body size, and both species are affected by genotoxic cell damage. This demonstrates that imidacloprid is potentially toxic for the two study species at environmentally relevant concentrations.

Swimming activity. Exposure to imidacloprid caused changes in tadpole swimming activity in comparison with the control in L. luctator only (Supplementary Table S3 Online). The most frequent behavioral alteration was lethargy (30.7% of the exposed tadpoles), followed by hyperactivity (20.7%), and spasms (18.7%). Almost a fifth (18%) of the treated tadpoles were unresponsive (Fig. 4).

Discussion
Exposure to imidacloprid caused morphological and genotoxic changes in the tadpoles of both L. luctator and P. cuvieri, although it did not affect survival. This was expected from the concentrations used in the present study (3-300 µg L −1 ), which were mostly below the LC 50 thresholds (82-366 mg L −1 ) reported for other amphibian species (reviewed by Gibbons et al. 44 ). The imidacloprid concentrations used in this study may nevertheless be representative of the levels of contamination found typically in surface water in agricultural areas 45 , and appear not to cause mortality but showed different chronic effects in other aquatic vertebrate species, such as R. sylvatica, exposed to 10-500 µg L −1 (cited as ppm) of the insecticide 43 , and the fish Pimephales promelas (10 µg L−1) 46 .
Changes in the development of the tadpoles were manifested by the reduced length and body mass observed in exposed tadpoles of both species, with L. luctator being more sensitive to imidacloprid than P. cuvieri. Under stress, such as the presence of contaminants, efforts to tolerate the presence of pesticides may compromise an individual's metabolism and growth 47 . Reduced development of the tadpoles may make them more vulnerable to predation in natural environments, because of both their smaller size (e.g., Carlson and Langkilde 48 ) and their reduced physical capacity. Although body size is a highly variable characteristic, there is a general correlation between the size of the tadpoles and the adults 49 . Previous studies have shown that tadpoles with reduced body size may develop into smaller adults, with lower rates of survival and reproductive success 50 . Leptodactylus luctator (denominated L. latrans in some previous studies) is a relatively large-bodied amphibian, which is important www.nature.com/scientificreports/ for the defense of the eggs and tadpoles, as well as the avoidance of predation 51 . Morphological changes in the tadpoles might also alter their eventual reproductive success, given that smaller females of both study species are known to be less fecund [52][53][54] . Increasing concentrations of imidacloprid caused malformations of the oral and intestinal structures of the tadpoles. The malformation of oral structures may restrict the growth of the individual and differences in tadpoles' oral morphology may affect its capacity to acquire food 55,56 . The oral structures of these tadpoles consist of labial teeth that are used as food scrapers, and changes in these structures may affect the ability of the tadpoles to forage 55,57,58 . Inefficient feeding may impact growth rates and the accumulation of body mass 57,59,60 , as well    Physalaemus cuvieri www.nature.com/scientificreports/ as increase the susceptibility of the individual to predation 55,61 . The structural integrity of the intestine is also important to guarantee efficient nutrient absorption and growth 62,63 .
Malformed individuals generally constitute a small proportion of natural amphibian populations, typically less than 2% 64 . In the present study, however, more than 50% of the individuals presented morphological malformations, reflecting the toxic effects of this compound on both study species. This neonicotinoid pesticide also caused morphological malformations in birds exposed to 2.5-20 µg 65 , and fish exposed to 300 µg L −1 and 1000 µg L −1 of imidacloprid 33 , reflecting the more ample potential environmental impacts of this this insecticide.
Despite the alterations observed in the morphology of the tadpoles of both study species, only L. luctator presented changes in swimming activity after exposure to imidacloprid. In addition to the dietary changes resulting from the malformations of the oral structures and the intestine, a reduced food intake due to lethargy or increased energetic expenditure in individuals with hyperactivity may have contributed to the reduced growth and greater sensitivity of L. luctator to imidacloprid. Alterations of swimming activity have also been observed in other amphibians 13,35 , and fish 66 exposed to imidacloprid, and a decrease in spontaneous locomotor activity has been recorded in rats 67 , possibly due to the neurobehavioral impacts of this pesticide 66 .
Genotoxic alterations were also observed in the L. luctator and P. cuvieri tadpoles exposed to imidacloprid in the present study. This genotoxic response has been associated with the increased production of reactive oxygen species by the pesticides, which promotes oxidative stress that inhibits the activity of the enzymes involved in DNA repair. This causes the formation of micronuclei and other nuclear abnormalities 68,69 . The frequency of all ENAs, particularly notched and lobed nuclei, increased considerably at imidacloprid concentrations of 30 µg L −1 and over, demonstrating the extreme genotoxic potential of this pesticide. Nuclear abnormalities are considered to be biomarkers of the impact of pesticides on amphibians 14,70,71 . As in the present study, notched nuclei were the most common abnormality in Boana pulchella tadpoles exposed to a pirimicarb-based compound 72 . Any external factor that affects cell proliferation, differentiation or apoptosis can produce embryotoxic or teratogenic effects, and may result in permanent congenital malformations, functional abnormalities or even the death of the individual 73 .
Micronuclei, APs, NBs, and BCs all reached significant levels in both L. luctator and P. cuvieri, in particular at the highest imidacloprid concentrations. The formation of micronuclei is related to failures in mitotic division 74 and may be triggered by the presence of nuclei with bubbles or cellular binucleation 75 , which results from the blockage of cytokinesis by abnormal cell division (reviewed by Benvindo-Souza et al. 76 ). Apoptotic nuclei undergo nuclear disintegration without suffering any alteration of the cytoplasm 77 , which indicates cell death 76 and is often associated with neurological disorders 78 . Exposure to higher concentrations of imidacloprid than those tested in the present study revealed genotoxic effects in the amphibians Rana sp. (exposed to 0.05 mg L −1 , 0.5 mg L −1 , 8 mg L −1 , and 32 mg L −1 of imidacloprid) 37 , and Boana pulchella (15 mg L −1 ) 38 , as well as in the fish Australoheros facetus (100 µg L −1 and 1000 µg L −1 ) 79 , and Prochilodus lineatus (up to 1250 µg L −1 ) 80 . It is important to note that, due to the scarcity of data on the concentration of pesticides in the environment, the first studies were based on doses lower than those recommended for agricultural crops, although they were still high in comparison with the subsequent studies. The accumulation of data on both the amount of pesticides in the environment [40][41][42][81][82][83] and their ecotoxicological effects on amphibians, has allowed the most recent studies to apply more realistic doses.
Based on the alterations in swimming activity and the morphological malformations and genotoxicity observed in the present study, we can conclude that 3 μg L −1 of imidacloprid can cause chronic effects for both L. luctator and P. cuvieri. At higher concentrations, both species are likely to present morphological malformations, and L. luctator may also exhibit reduced growth and altered swimming activity. At concentrations of over 3 μg L −1 , P. cuvieri presents reduced growth, and the tadpoles of both species had genotoxic cell damage. The fact that the short-term assay used in the present study was sufficient to cause cytotoxic damage to the tadpole cells indicates just how potentially toxic this pesticide is to amphibians. Detectable concentrations of imidacloprid in water range from 0.001 to 320 μg L −1 , although the mean maximum concentration in surface water is 18.65 μg L −1 (n = 21 studies; see Morrissey et al. 45 ). This emphasizes the need for more research that focuses on limiting the concentrations of this pesticide at different trophic levels, and the importance of updating existing legislation to protect aquatic wildlife.
Although L. luctator and P. cuvieri belong to the same family (Leptodactylidae), are widely distributed in South America, and are adapted to a range of different habitats 10,84 , L. luctator was more sensitive to exposure to imidacloprid, and this is the first study to highlight this difference. A previous study 85 also found differences in the sensitivity of the amphibians Acris crepitans and Rana clamitans to imidacloprid, which is an important consideration when selecting species as bioindicators, given that they need to be both sensitive to contaminants and abundant enough for systematic monitoring.
As tadpoles develop in a liquid medium, they are unable to escape exposure to contamination in aquatic environments, which may reduce their capacity to reach an adequate level of development to survive in the terrestrial environment. Neonicotinoid compounds such as imidacloprid are not only highly toxic but also persistent [86][87][88] , and impact distinct trophic levels in aquatic environments by reducing body mass, which alters the dynamics of the food chain, especially for the top-level consumers 89 . Given this, we emphasize the importance of the implementation of effective conservation measures, associated with the review or creation of specific legislation that will mediate the impact of pesticides on wild populations of anuran amphibians. The clear evidence of the toxic effects of imidacloprid on anuran amphibians and the extensive and unregulated agricultural use of other neonicotinoids in Brazil and worldwide highlights the need for further, more systematic research to better assess the risks of the use of these pesticides for anuran populations.

Conclusion
We found that environmentally relevant concentrations of the neonicotinoid insecticide imidacloprid induced significant alterations in the development of the tadpoles of L. luctator and P. cuvieri. Significant morphological and genotoxic alterations were observed in both species, although L. luctator was more sensitive to the insecticide than P. cuvieri. Even the lowest tested concentration of the insecticide (3 μg L −1 ) was harmful to amphibians, a concentration 100 times lower than that permitted by environmental legislation in Brazil.

Methods
Tadpole species. Spawn of L. luctator and P. cuvieri were collected within 24 h of oviposition from nonagricultural land with no known use of pesticides in Erechim, Rio Grande do Sul state, Brazil (27°42′43.77″ S, 52°18′42.94″ W). The spawn was placed immediately in aquariums containing 15 L of dechlorinated water at the Ecology and Conservation Laboratory of the Erechim campus of the Federal University of Fronteira Sul. The eggs were raised under controlled conditions of temperature (24 ± 2 °C) and light (12/12 h light/dark photoperiod) until they reached development stage 25 90 . The water was monitored daily and presented the following parameters: pH = 7.5 ± 0.5, dissolved oxygen = 5.8 ± 0.4 mg L −1 , turbidity = < 5, conductivity = 649 ± 25 µS cm −1 , hardness = 3.57 mg L −1 , Na = 13.012 mg L −1 , and Ni = < 0.002 mg L −1 . The tadpoles were fed daily with complete fish feed (Alcon Basic, Alcon) containing at least 45% crude protein and organic lettuce. This study was approved by the Ethics Committee for the Use of Animals (CEUA) of the Fronteira Sul Federal University under protocols nº 8822130919 and nº 8742250320, and was authorized by the Chico Mendes Institute for Biodiversity Conservation (ICMBio) under license nº 72719. All methods were carried out in accordance with relevant guidelines and regulations, and as reported by the ARRIVE guidelines 91 .
Experimental design and experimental conditions. The tadpoles in development stage 25 used in the tests had completely-formed oral structures, normal swimming activity, and had typical, homogeneous body length and mass. The tadpoles of L. luctator had a mean length of 13.25 ± 0.36 mm and body mass of 0.035 ± 0.008 g, while those of P. cuvieri had means of 16.60 mm ± 0.60 mm and 0.070 g ± 0.011 g.
These tadpoles were exposed to the insecticide in a static test over a standard period of 168 h (7 days) according to ASTM STP 1443 92 , during which, they were fed daily as described above. The tadpoles were exposed to five water treatments defined by the following nominal concentrations of imidacloprid (48% a.i., Imidacloprid Nortox, Nortox S/A, Arapongas, Brazil) added to the water of the aquarium: (i) 3 μg a.i. L −1 , (ii) 30 μg a.i. L −1 ; (iii) 100 μg a.i. L −1 ; (iv) 200 μg a.i. L −1 , and (v) 300 μg a.i. L −1 , together with a control treatment, containing clean water only. The experiments were run with a randomized block design. Batches of 10 tadpoles were transferred to 500 mL glass containers, with each container being considered as an experimental unit. The assays were conducted in triplicate, with a total of 30 tadpoles per treatment. The physical-chemical characteristics of the water were the same as those used for the development of the tadpoles, with ammonia being measured daily (mean = 0.283 ± 0.038 mg L −1 ).
The pesticide concentrations were selected based on the imidacloprid value recorded in the surface water in Brazil (3 μg L −1 ) 40,41 and in rice paddies in Vietnam (30 μg L −1 ) 42 , as well as the legal limit established in the Brazilian state of Rio Grande do Sul (300 μg L −1 ) 31 , and two intermediate concentrations. Merga and Van den Brink 93 reported that the imidacloprid-based insecticide used in the present study remained at a constant concentration throughout their 96-h experiment. While the period of the present study was three days longer than this, and photolysis is known to degrade imidacloprid when luminosity or temperatures are high 94,95 , the conditions of the study were adequate to minimize either dissipation or degradation.
Survival, swimming activity and body size and morphological malformations. Tadpole survival was verified every 24 h, when the number of live and dead tadpoles in each container was recorded. The dead tadpoles were removed from the containers. Swimming activity was also recorded every 24 h by qualitative observation, based on Rutkoski et al. 70 , with modifications. The tadpoles were stimulated gently with a glass rod and the response was recorded. For this, all the tadpoles in a given container were observed qualitatively at the same time by the same observer. Qualitative changes in behavior were assessed during the course of the experimental exposure by observing changes in the response of the tadpoles over a 1 min interval, using of a behavioral checklist, similar to that recommended for fish by ASTM E1711-12, to document the response of the animals. The activity of the tadpoles was classified as: (i) swimming activity equal to the control, (ii) lethargy (reduced swimming activity in comparison with the control), (iii) hyperactivity (increased swimming activity in comparison with the control), (iv) unresponsive (no movement), and (v) spasms (tremors and convulsions).
At the end of the assay period, the tadpoles were euthanized with lidocaine (5%) following the rules of the Brazilian National Council for Animal Control and Experimentation 96 . The total length (mm; snout to tail) and body mass (g) of these tadpoles were measured using digital calipers and a precision balance, respectively. Malformations of the oral structures (denticles or general morphology) and the intestine (edemas or general morphology) were evaluated according to Rutkoski et al. 70,97 . Digital images of the oral and intestine structures were obtained using a digital camera (P510, Nikon, Tokyo, Japan) and analyzed in comparison with the control, using a stereomicroscope (SZ51, Olympus, Tokyo, Japan).
Micronucleus assay and other erythrocytic nuclear abnormalities. For genotoxic analysis, a drop of blood obtained from each of the 10 tadpoles selected randomly from each treatment was placed on slides and fixed and stained with Panotic Rapid stain (Laborclin Ltda, Brazil), according to the manufacturer's instructions. The slides were analyzed under an optical microscope with a 100 × lens (CX31, Olympus, Tokyo, Japan), with 1000 cells being examined from each individual. The cells were examined for the presence of erythrocyte nuclear Scientific Reports | (2022) 12:11926 | https://doi.org/10.1038/s41598-022-16039-z www.nature.com/scientificreports/ abnormalities (ENAs), including micronuclei (MN). The micronuclei were analyzed following the protocol of Pérez-Iglesias et al. 98 , while the other six ENAs were analyzed according to Montalvão et al. 99 , being classified as apoptosis (AP), binucleated cells (BC), karyolysis (KA), lobed nuclei (LN), nuclear bubbles or buds (NB), and notched nuclei (NN).

Statistical analyses.
The normality and variance homogeneity of the data were confirmed using the Kolmogorov-Smirnov and Barlett tests, respectively. A one-way analysis of variance (ANOVA) was applied to the data on survival, body size, morphological malformations, swimming activity, MNs, and ENAs. Pairwise comparisons between each treatment and the control were based on Dunnett's test (p < 0.05). The statistical analyses were performed in Statistic 8.0, and the graphs were produced in GraphPad Prism 7.0.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).