Study area and specimen collection
The present study was carried out in two areas of the Brazilian Cerrado biome that have distinct histories of land use. The agricultural environment selected for the study was located in the municipality of Rio Verde, in southwestern Goiás, a state in Brazil’s Midwest region. This municipality is considered to be the state’s principal producer of soybean (IMB 2019) and prior to 2017, it ranked eighth in the national ranking of soybean exporting municipalities (IBGE 2017). Rio Verde thus represents an environment with a high probability of genotoxic impacts on the resident organisms (Borges et al. 2019b). In this environment, 10 adult male Leptodactylus fuscus were collected on a single night in November 2018, during the soybean planting period, through an active nocturnal search. The animals were all collected in the vicinity of a single temporary pond of approximately 2m x 5m, and 15cm in depth, located in the middle of a soybean plantation (17º47.742’ S, 051º06.089’ W). There are sparse fragments of forest in the surrounding area, but no other bodies of water nearby, and there is vegetation in the water. Data obtained from the NASA Prediction of Worldwide Energy Resources (NASA POWER) website (https://power.larc.nasa.gov/#dataaccess) show that the mean minimum temperature of this point in 2018 was approximately 17.86°C, the maximum temperature was almost 28.77°C, precipitation was 3.51 mm/day, and the incidence of sunlight was 5.11 Kw/h/m2/day.
Another 10 adult male L. fuscus were collected by active night searching in the Emas National Park (ENP) in December 2018. The ENP, a federal conservation unit with an area of approximately 132,000 hectares, is located in southwestern Goiás, in the municipalities of Mineiros and Chapadão do Céu, and in the neighbouring area of the state of Mato Grosso do Sul, in the municipality of Costa Rica (ICMBio 2019). This area is relatively flat, and encompasses different Cerrado formations, such as grassland, shrubby savanna, and riparian forests (ICMBio 2019). In this environment, the specimens were collected on a single night from a large humid area close to a body of water located at least 10 km from the outer perimeter of the national park (18º06.990’ S, 052º55.024’ W). At the collection site, there is a predominance of typical Cerrado shrubby savanna vegetation, with sparse and poorly developed shrubs and herbaceous plants (ICMBio 2019). The mean minimum temperature recorded for the location in 2018 was approximately 18.18ºC, the mean maximum temperature was around 29.29°C, mean precipitation was 3.15 mm/day and insolation was 5.10 Kw/h/m2/day (data obtained from the NASA POWER website).
All the specimens were taken to the Animal Biology Laboratory of the Goiano Federal Institute in Rio Verde, where the analyses were conducted. The total weight each specimen was determined using a precision analytical balance and the snout-vent length was recorded using a digital caliper (0.01 mm precision). The characteristics of the matrix surrounding each sampling point were recorded within a radius of 1 km (Fig. 1). The ENP is covered (100%) in natural vegetation, with grassland and savannah typical of the Cerrado biome, while the soybean plantation is dominated by crops (92.59%), with only 7.41% of natural vegetation cover.
Ethical and sampling statement
For this study, licenses for the collection of specimens and animal experimentation were obtained from the Chico Mendes Institute for Biodiversity Conservation (ICMBio), under protocol 62687-1, and the Goiano Federal Institute Ethics Committee on the Use of Animals (CEUA/IFGoiano) under protocol number 6643030518.
Micronuclei and other erythrocyte nuclear abnormalities
After collection, the animals were euthanized by immersion in the anesthetic Benzocaine (5g/L). An abdominal incision was then made, from which the circulating blood cells of the abdominal cavity were obtained with the aid of a heparinized needle and syringe (25 mm x 0.7 mm). Two blood slides were prepared per animal, which were fixed in cold methanol for 20 minutes and then stained with Giemsa solution (5%) in tap water for 12 minutes (Vera-Candioti 2010). A total of 2000 cells per animal were analyzed under an optical microscope (Laborana LAB-1001TB) attached to a digital camera (Laborana 3.0Mp), using 100x magnification (Cruz-Esquivel et al. 2017).
The criteria used to identify micronuclei were (a) staining intensity equal to that of the principal nucleus of the cell, but with a smaller diameter, (b) rounded shape unconnected to the principal nucleus, and (c) no overlap with the principal nucleus and located within the cytoplasm (Fenech 2000). The other ENAs quantified in the analyses were binucleated cells, nuclear buds, anucleated cells, lobed nuclei, notched nuclei, reniform (or kidney shaped) nuclei, and segmented nuclei, as described in our recent review (Benvindo-Souza et al. 2020). The data were presented as standard frequencies for each anomaly and for the whole set of ENAs (Pollo et al. 2015; Borges et al. 2019b).
Hepatic melanin
For the histological analyses, liver fragments were extracted from each L. fuscus specimen, weighed on a precision analytical balance, and fixed in metacarn (60% methanol, 30% chloroform, and 10% acetic acid) for 3 hours at 4ºC. These samples were then dehydrated in an increasing alcohol series and embedded in historesin (Leica-historesin embedding kit) for the extraction of 2µm sections using a Leica RM 2265 microtome (Switzerland). These sections were placed on slides, stained with Hematoxylin-Eosin (HE), and photographed under a Leica DM4 B microscope with a 20x magnification attached to an image capture system (Leica DMC 4500). Hepatic pigmentation was quantified based on color intensity, using the Image Pro-Plus Media-Cybernetics Inc. program (version 6.0). A total of 25 random histological fields were photographed and analyzed in each specimen. The procedures followed the protocols of Santos et al. (2014), Franco-Belussi et al. (2016), and Fanali et al. (2018).
Water quality
Water samples were collected from both study sites (Rio Verde and the ENP) for physical-chemical analyses. The samples were collected in Rio Verde in November 2018 at approximately 20:00 h and in the ENP in December 2018 at the same time, and were then sent to the laboratory of analysis within 24 hours of collection. One liter of water was collected from approximately 5 cm below the water surface of a single pond close to the study site and on the same day that the animals were collected in each environment. The samples were stored in individual amber borosilicate glass flasks at a temperature of <4ºC and sent to the Germinar Agroanálises & Ambiental laboratory in Rio Verde, Goiás, Brazil, for the quantification of carbamate, organochlorine, and organophosphate pesticides. The following substances were analyzed: 2. 4-D + 2. 4. 5-T, Alachlor; Aldicarb + Aldicarb-sulfone + Aldicarb-sulfoxide, Aldrin + Dieldrin, Atrazine, Carbendazim + Benomyl, Carbofuran; Chlordane (Cis + Trans), Chlorpyrifos + Chlorpyrifos-Oxon, DDDT, Diuron, Endosulfan (Alpha + Beta + Sulfate), Endrin, Glyphosate + Ampa, Lindane, Mancozeb, Metamidophos, Metolachlor, Molinate, Methyl Paration, Pendimethalin, Permethrin, Profenofos, Simazine, Tebuconazole, Terbufos, and Trifluralin. All of these compounds can be found in agricultural areas, either after recent application or in residual form from previous land use. The analyses were conducted according to the procedures outlined in Standard Methods for the Examination of Water and Wastewater, 23rd edition. The parameters were assessed based on the standards defined by CONAMA resolution number 357/2005 - Class II and decree 1,745/1979.
The temperature, pH, dissolved oxygen, conductivity, resistivity, salinity, and total dissolved solids of the water of each study pond were measured in situ using a portable multi-parameter Bante900P apparatus. These measurements were conducted on the day of the collection of the animal specimens at each site, at approximately 20:00h.
Statistical analyses
The MN and ENA data, and the melanin scores are presented as means±standard deviation. The MN and ENA frequencies and the area of melanin were compared systematically between the two study areas (soybean plantation and the ENP). Prior to the analyses, the homogeneity of the variances was evaluated using Levene’s test, and the data were (Log10) transformed to homogenize the variances, as appropriate. Student’s t (Pollo et al. 2017) was applied to the parametric data (micronuclei, segmented nucleus, reniform nuclei, notched nuclei, anucleated cells, nuclear buds, binucleated cells, total ENAs, and the area of hepatic melanin), while Mann Whitney’s U was applied to the nonparametric data (lobed nuclei). A multiple Pearson correlation analysis was applied to verify the existence of a correlation between the frequency of MNs, other ENAs, and the area of hepatic melanin in each environment. A p<0.05 value was considered significant in all analyses.