This study assessed the lethality, sublethality, and toxicity of commercial cypermethrin formulations to anuran tadpoles. In the acute test, concentrations of 100–800 μg/L were tested for 96 h. In the chronic test, naturally occurring cypermethrin concentrations (1, 3, 6, and 20 μg/L) were tested for mortality, followed by micronucleus testing and red blood cell nuclear abnormalities for 7 days. The LC50 of the commercial cypermethrin formulation to tadpoles was 273.41 μg L−1. In the chronic test, the highest concentration (20 μg L−1) resulted in greater than 50% mortality, as it killed half of the tadpoles tested. The micronucleus test showed significant results at 6 and 20 μg L−1 and several nuclear abnormalities were detected, indicating that the commercial cypermethrin formulation has genotoxic potential against P. gracilis. Cypermethrin is a high risk for this species, indicating that it can cause multiple problems and affect the dynamics of this ecosystem in the short and long term. Therefore, it can be concluded that commercial cypermethrin formulations have toxic effects on P. gracilis.
Due to the continuous expansion of agricultural activities and intensive application of pest control measures, aquatic animals are frequently exposed to pesticides1,2. Pollution of water resources near agricultural fields can affect the development and survival of non-target organisms such as amphibians.
Amphibians are becoming increasingly important in the assessment of environmental matrices. Anurans are considered good bioindicators of environmental pollutants due to their unique characteristics such as complex life cycles, rapid larval growth rates, trophic status, permeable skin10,11, dependence on water for reproduction12 and unprotected eggs11,13,14. The little water frog (Physalaemus gracilis), commonly known as the weeping frog, has been shown to be a bioindicator species of pesticide pollution4,5,6,7,15. The species is found in standing waters, protected areas or areas with variable habitat in Argentina, Uruguay, Paraguay and Brazil1617 and is considered stable by the IUCN classification due to its wide distribution and tolerance of different habitats18.
Sublethal effects have been reported in amphibians following exposure to cypermethrin, including behavioural, morphological and biochemical changes in tadpoles23,24,25, altered mortality and metamorphosis time, enzymatic changes, decreased hatching success24,25, hyperactivity26, inhibition of cholinesterase activity27 and changes in swimming performance7,28. However, studies of the genotoxic effects of cypermethrin in amphibians are limited. Therefore, it is important to assess the susceptibility of anuran species to cypermethrin.
Environmental pollution affects the normal growth and development of amphibians, but the most serious adverse effect is genetic damage to DNA caused by pesticide exposure13. Blood cell morphology analysis is an important bioindicator of pollution and potential toxicity of a substance to wild species29. The micronucleus test is one of the most commonly used methods for determining the genotoxicity of chemicals in the environment30. It is a rapid, effective and inexpensive method that is a good indicator of chemical pollution of organisms such as amphibians31,32 and can provide information on exposure to genotoxic pollutants33.
The objective of this study was to evaluate the toxic potential of commercial cypermethrin formulations to small aquatic tadpoles using a micronucleus test and ecological risk assessment.
Cumulative mortality (%) of P. gracilis tadpoles exposed to different concentrations of commercial cypermethrin during the acute period of the test.
Cumulative mortality (%) of P. gracilis tadpoles exposed to different concentrations of commercial cypermethrin during a chronic test.
The observed high mortality was a result of genotoxic effects in amphibians exposed to different concentrations of cypermethrin (6 and 20 μg/L), as evidenced by the presence of micronuclei (MN) and nuclear abnormalities in erythrocytes. Formation of MN indicates errors in mitosis and is associated with poor binding of chromosomes to microtubules, defects in protein complexes responsible for chromosome uptake and transport, errors in chromosome segregation and errors in DNA damage repair38,39 and may be related to pesticide-induced oxidative stress40,41. Other abnormalities were observed at all concentrations evaluated. Increasing cypermethrin concentrations increased nuclear abnormalities in erythrocytes by 5% and 20% at the lowest (1 μg/L) and highest (20 μg/L) doses, respectively. For example, changes in the DNA of a species can have serious consequences for both short- and long-term survival, resulting in population decline, altered reproductive fitness, inbreeding, loss of genetic diversity, and altered migration rates. All of these factors can impact species survival and maintenance42,43. The formation of erythroid abnormalities may indicate a block in cytokinesis, resulting in abnormal cell division (binucleated erythrocytes)44,45; multilobed nuclei are protrusions of the nuclear membrane with multiple lobes46, while other erythroid abnormalities may be associated with DNA amplification, such as nuclear kidneys/blebs47. The presence of anucleated erythrocytes may indicate impaired oxygen transport, especially in contaminated water48,49. Apoptosis indicates cell death50.
Other studies have also demonstrated the genotoxic effects of cypermethrin. Kabaña et al.51 demonstrated the presence of micronuclei and nuclear changes such as binucleated cells and apoptotic cells in Odontophrynus americanus cells after exposure to high concentrations of cypermethrin (5000 and 10,000 μg L−1) for 96 h. Cypermethrin-induced apoptosis was also detected in P. biligonigerus52 and Rhinella arenarum53. These results suggest that cypermethrin has genotoxic effects on a range of aquatic organisms and that the MN and ENA assay may be an indicator of sublethal effects on amphibians and may be applicable to native species and wild populations exposed to toxicants12.
Commercial formulations of cypermethrin pose a high environmental hazard (both acute and chronic), with HQs exceeding the US Environmental Protection Agency (EPA) level54 that may adversely affect the species if present in the environment. In the chronic risk assessment, the NOEC for mortality was 3 μg L−1, confirming that the concentrations found in water may pose a risk to the species55. The lethal NOEC for R. arenarum larvae exposed to a mixture of endosulfan and cypermethrin was 500 μg L−1 after 168 h; this value decreased to 0.0005 μg L−1 after 336 h. The authors show that the longer the exposure, the lower the concentrations that are harmful to the species. It is also important to highlight that the NOEC values were higher than those of P. gracilis at the same exposure time, indicating that the species response to cypermethrin is species-specific. Furthermore, in terms of mortality, the CHQ value of P. gracilis after exposure to cypermethrin reached 64.67, which is higher than the reference value set by the US Environmental Protection Agency54, and the CHQ value of R. arenarum larvae was also higher than this value (CHQ > 388.00 after 336 h), indicating that the studied insecticides pose a high risk to several amphibian species. Considering that P. gracilis requires approximately 30 days to complete metamorphosis56, it can be concluded that the studied concentrations of cypermethrin may contribute to population decline by preventing infected individuals from entering the adult or reproductive stage at an early age.
In the calculated risk assessment of micronuclei and other erythrocyte nuclear abnormalities, the CHQ values ranged from 14.92 to 97.00, indicating that cypermethrin had a potential genotoxic risk to P. gracilis even in its natural habitat. Taking into account mortality, the maximum concentration of xenobiotic compounds tolerable to P. gracilis was 4.24 μg L−1. However, concentrations as low as 1 μg/L also showed genotoxic effects. This fact may lead to an increase in the number of abnormal individuals57 and affect the development and reproduction of species in their habitats, leading to a decline in amphibian populations.
Commercial formulations of the insecticide cypermethrin showed high acute and chronic toxicity to P. gracilis. Higher mortality rates were observed, likely due to toxic effects, as evidenced by the presence of micronuclei and erythrocyte nuclear abnormalities, especially serrated nuclei, lobed nuclei, and vesicular nuclei. In addition, the studied species showed increased environmental risks, both acute and chronic. These data, combined with previous studies by our research group, showed that even different commercial formulations of cypermethrin still caused decreased acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities and oxidative stress58, and resulted in changes in swimming activity and oral malformations59 in P. gracilis, indicating that commercial formulations of cypermethrin have high lethal and sublethal toxicity to this species. Hartmann et al. 60 found that commercial formulations of cypermethrin were the most toxic to P. gracilis and another species of the same genus (P. cuvieri) compared with nine other pesticides. This suggests that legally approved concentrations of cypermethrin for environmental protection may result in high mortality and long-term population decline.
Further studies are needed to assess the toxicity of the pesticide to amphibians, as the concentrations found in the environment may cause high mortality and pose a potential risk to P. gracilis. Research on amphibian species should be encouraged, as data on these organisms are scarce, particularly on Brazilian species.
The chronic toxicity test lasted for 168 h (7 days) under static conditions and the sublethal concentrations were: 1, 3, 6 and 20 μg a.i. L−1. In both experiments, 10 tadpoles per treatment group were evaluated with six replicates, for a total of 60 tadpoles per concentration. Meanwhile, the water-only treatment served as a negative control. Each experimental setup consisted of a sterile glass dish with a capacity of 500 ml and a density of 1 tadpole per 50 ml of solution. The flask was covered with polyethylene film to prevent evaporation and was continuously aerated.
The water was chemically analyzed to determine pesticide concentrations at 0, 96, and 168 h. According to Sabin et al. 68 and Martins et al. 69 , the analyses were performed at the Pesticide Analysis Laboratory (LARP) of the Federal University of Santa Maria using gas chromatography coupled to triple quadrupole mass spectrometry (Varian model 1200, Palo Alto, California, USA). The quantitative determination of pesticides in water is shown as supplementary material (Table SM1).
For the micronucleus test (MNT) and red cell nuclear abnormality test (RNA), 15 tadpoles from each treatment group were analyzed. Tadpoles were anesthetized with 5% lidocaine (50 mg g-170) and blood samples were collected by cardiac puncture using disposable heparinized syringes. Blood smears were prepared on sterile microscope slides, air dried, fixed with 100% methanol (4 °C) for 2 min, and then stained with 10% Giemsa solution for 15 min in the dark. At the end of the process, slides were washed with distilled water to remove excess stain and dried at room temperature.
At least 1000 RBCs from each tadpole were analyzed using a 100× microscope with a 71 objective to determine the presence of MN and ENA. A total of 75,796 RBCs from tadpoles were evaluated considering cypermethrin concentrations and controls. Genotoxicity was analyzed according to the method of Carrasco et al. and Fenech et al.38,72 by determining the frequency of the following nuclear lesions: (1) anucleate cells: cells without nuclei; (2) apoptotic cells: nuclear fragmentation, programmed cell death; (3) binucleate cells: cells with two nuclei; (4) nuclear buds or bleb cells: cells with nuclei with small protrusions of the nuclear membrane, blebs similar in size to micronuclei; (5) karyolyzed cells: cells with only the outline of the nucleus without internal material; (6) notched cells: cells with nuclei with obvious cracks or notches in their shape, also called kidney-shaped nuclei; (7) lobulated cells: cells with nuclear protrusions larger than the aforementioned vesicles; and (8) microcells: cells with condensed nuclei and reduced cytoplasm. The changes were compared with the negative control results.
The acute toxicity test results (LC50) were analyzed using GBasic software and the TSK-Trimmed Spearman-Karber method74. The chronic test data were pre-tested for error normality (Shapiro-Wilks) and homogeneity of variance (Bartlett). The results were analyzed using one-way analysis of variance (ANOVA). Tukey’s test was used to compare data among themselves, and Dunnett’s test was used to compare data between the treatment group and the negative control group.
LOEC and NOEC data were analyzed using Dunnett’s test. Statistical tests were performed using Statistica 8.0 software (StatSoft) with a significance level of 95% (p < 0.05).
Post time: Mar-13-2025