2.1 Chemical reagents and determinations
All chemicals, reactives and solvents of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO) while Pivot H® (10.59% w/v of IMZT, CAS 081335-77-5) was obtained from BASF Argentina S. A. Cyclophosphamide (CP, CAS 6055-19-2) was purchased from Sigma Chemical Co. (St. Louis, MO). IMZT concentrations present in bioassay solutions were analyzed by Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system coupled to a Quattro Premier XE tandem quadrupole mass spectrometer (MS/MS), with an electrospray ionization (ESI) source equipment from the CIM Institute (National University of La Plata, La Plata, Argentina) according to the procedures described in Report 01-4134 of the U.S. Geological Survey (Furlong et al., 2001). Samples from test nominal concentrations were taken and immediately measured after the solutions were prepared (0 h) and 24 h thereafter. Detection limit of IMZT was 0.5 µg/L.
2.2 Test organisms and specimen recollection
Boana pulchella (Duméril and Bibron, 1841), formerly named Hypsiboas pulchellus, is an arboreal anuran species from the Hylidae family with extensive distribution in neotropical South America, and which is very abundant in the Pampa region of Argentina (Cei, 1980). Its natural habitats are dry low land grasslands, seasonally wet or flooded low land grasslands, intermittent fresh water lakes, intermittent fresh water marshes, and pasture lands (Kwet et al., 2004). This species lays its eggs in masses attached to the submerged stems of aquatic plants. This species is easy to acclimate to laboratory conditions, it has been previously employed as a bioindicator species (Lajmanovich et al., 2005; Agostini et al. 2009; Brodeur et al. 2011; 2012; Pérez-Iglesias et al., 2014; 2015; Brodeur and Vera Candioti, 2017; 2018; Sansiñena et al. 2018) and its population status is considered as “least concern” (Vaira et al., 2012; IUCN, 2020). Individuals of B. pulchella (n = 100) were obtained during night collections from a typical pampean unpolluted habitat, during breeding season (La Plata, Buenos Aires, Argentina − 35° 10′ S; 57° 51′ W). All frog collections were approved by the Buenos Aires Province government (permit number 22500 − 22339/13). All experimental procedures were performed according to “Reference Ethical Framework for Biomedical Research: Ethical Principles for Research with Laboratory, Farm, and Wild Animals” (CONICET, 2005). Adult males of B. pulchella were transported to the laboratory to be weighted (average, 2.65 ± 0.52 g) and measured (snout-vent length average, 35.35 ± 2.58 mm). Afterwards, specimens were acclimated during 7 days in 2800 cm3 glass containers containing 200 cm3 of soil at the bottom. Photoperiod (16:8 h light/dark) and temperature (25.0 ± 1°C) were maintained constant and animals were not fed during this period.
2.3 Experimental design and exposure protocol
Experiments were carried out reproducing plausible three exposure scenarios as recommended by Wang and Jia (2009) and Van Meter et al. (2015). The first scenario (S1) illustrated an exposure to field runoff water. The second scenario (S2) illustrated a situation where B. pulchella visited a leaf previously sprayed with a foliar application and in which one-tenth (1/10) of the applied concentration of IMZT would reach the frogs (100 mg IMZT/L) and finally, the third scenario was designed to illustrate a situation where frogs were directly sprayed with the recommended application rate (S3: 1000 mg IMZT/L). Briefly, before the start of the bioassay, frogs were placed in a clean 20 L glass aquarium for a 24 h period of dehydration. This dehydration period was intended to facilitate the movement of water and xenobiotics throughout the anuran dermis because the re-hydration would occur during the bioassay, according to Van Meter et al. (2018). For each scenario, adults’ frogs were exposed to an acute pulse of the IMZT-based herbicide formulation by immersing the entire specimens for 15 sec in each herbicide concentrations (10, 100 and 1000 mg IMZT/L). Test solutions were prepared according to procedures proposed by U.S. EPA (1975). Also, negative control and positive groups were prepared consisting of 10 frogs immersed in dechlorinated tap water and cyclophosphamide, respectively; and run in parallel with herbicide-exposed specimens. Immediately after exposure, each frog was placed individually in 3 L glass flasks containing 200 mL of fertile soil at the bottom. Frogs were not fed throughout the experiment and were re-hydrated by spraying dechlorinated tap water every 24 h to avoid frogs’ death due to drying (Van Meter et al., 2018). Evaluation of the proposed endpoints was performed 48 and 96 h after the acute exposure pulse. At each sampling time, frogs were anesthetized, placed on ice and dissected according to directives and protocols detailed in the Guide for Care and Use of Laboratory Animals (Garber et al., 2011), and the ethical procedures of the Ethical Committee from the National University of La Plata (code11/N619), “Reference Ethical Framework for Biomedical Research” (CONICET, 2005) and “Guide for Care and Use of Experimental Animals” (INTA, 2008). Experiments were performed in triplicate and 10 individuals were employed each time.
2.4 Evaluations of endpoints
2.4.1 Individual endpoints
Frog body condition was assessed using a method described by Brodeur et al. (2011). This method consists in examining the residuals from a regression of body mass against snout-vent length where the regression line obtained establishes the average body weight for a given length. Then an individual with positive residuals considered being in a good condition whereas an individual with a negative residual regarded as having low energy (Schulte-Hostedde et al., 2005; Brodeur et al., 2011). To evaluate behavioral sublethal endpoints, the mobility and posture of the frogs was observed during 1 min in a polypropylene chamber (30 L). Frogs were then placed into a pool to examine swimming activity for another minute. Finally, the hepatosomatic index (HSI) was calculated as the ratio of liver weight with respect to total body weight. At the end of the experiment the liver was weighed to obtain the HSI index using a precision scale 0.001 g.
2.4.2 Biochemical endpoints
All procedures were performed as previously described by Brodeur et al. (2017; 2020). Briefly, livers were homogenized in ice-cold 50mM tris buffer (1 mM EDTA acid, 0.25 M of sucrose, pH 7.4) with a Teflon-glass Potter-Elvehjem homogenizer. Then, the homogenates were centrifuged at 4°C (10,000 x g, 10 min) to collect the supernatant while nuclei and cell debris was discarded. One portion of the supernatant was used for protein concentrations were determined by the method of Lowry (1951) using bovine serum albumin as a standard. All enzymatic reactions and protein calibration curve were performed on microplates, and posterior reads of the enzymatic activities as well as the protein concentration were carried out by using a microplate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany).
GST activity determination
The GST activity in the liver of B. pulchella was measured using 1-chloro-2, 4- dinitrobenzene (CDNB) as substrate. Determinations were performed in a reaction mixture containing 300 µL GST (30% m/v of GSH in PBS, pH 7), 10 µL CDNB (0.1 M) and 10 µL of sample (dilution, 1:25 of pure supernatant:PBS). The colorimetric reaction absorbance (340 nm) was recorded during 2 min (37°C) and GST activity was calculated with molar extinction coefficient of 9.6 mM− 1cm− 1.
AChE activity determination
Activity of hepatic acetylcholinesterase (AChE) was determined by the Ellman method (1961). The reaction mixture consisted of 200 µL of PBS (100 mM, pH 8), 10 µL of acetylcholine (1 mM), 10 µL of DTNB (0.5 mM), and 50 µl of sample (previously diluted 1/5; 200 µL homogenized sample in 800 µL of PBS). The kinetic absorbance (412 nm) was recorded during 3 min (37°C) and AChE activity was calculated using a molar extinction coefficient of 14,150 M− 1 cm− 1.
CAT activity determination
The CAT activity in the liver of B. pulchella was determined by measuring the kinetic absorbance in microplates making reaction mixture of 300 µL of PBS (100 mM, pH 7), 10 µL of H2O2 (dilution 0,5% v/v, H2O2 99% in distilled water) and 10 µL of sample (dilution, 1:25 of pure supernatant:PBS). The change in the absorbance (240 nm) resulting from H2O2 consumption was recorded during 2 min (37°C), using a molar extinction coefficient of 43.6 M− 1 cm− 1.
2.4.3 Cytogenetic endpoints
MNs induction and nuclear abnormalities
MN assay and blood analysis was conducted in accordance with original protocol (Fenech, 2007) with our minor modifications for this species (Pérez-Iglesias et al., 2016). Slides of blood smears, by triplicate, were stained during 12 min with 5% of Giemsa solution for each treated group. MNs frequency was calculated in peripheral mature erythrocytes after acute pulse exposure in both scenarios. MNs were blind-scored from 1000 erythrocytes from each blood frog sample (×1000 magnification). Besides, the presence of other nuclear abnormalities in mature erythrocytes was evaluated for this species according our previous procedures (Pérez-Iglesias et al., 2016; 2020). The following frequency of nuclear abnormalities were considered: notched nuclei (NNs), i.e. nuclei with vacuoles and appreciable depth into a nucleus without containing nuclear material; blebbed nuclei (BLs), i.e. cells with one nucleus presenting a relatively small evagination of the nuclear membrane which contains euchromatin; and erythroplastids (EPs), i.e. anucleated forms of circulating red blood cells. MNs and nuclear abnormalities frequencies are expressed as total number of alterations per 1000 cells and the examination criteria for MNs acceptance was determined following previously reports (Vera Candioti et al., 2010).
Comet assay
The same individuals employed for the MN assay (see Section 2.4.3.2) were also used for the comet assay. The comet assay was performed following the alkaline procedure described elsewhere for the species (Pérez-Iglesias et al. 2014; 2015; 2017; 2018). Briefly, blood samples were diluted in PBS, centrifuged (2,000 rpm, 9min), and resuspended in PBS (50 mL). An aliquot of diluted samples (30 mL) was mixed with low-melting-point agarose (70 mL, 0.5%) and was then layered on a slide precoated with normal-melting-point agarose (100 mL, 0.5%). The slide was placed at 4°C. After solidification, the slide was covered with a third layer of low-melting-point agarose (50 mL, 0.5%). After that, the slides were immersed in ice-cold freshly prepared lysis solution and then lysed in darkness for a 1h (4°C). Then, slides were placed in an electrophoresis buffer (4°C) to allow the cellular DNA to unwind, followed by electrophoresis in the same buffer (4°C, 20 min, 25 V). Finally, the slides were neutralized with a solution comprising Tris–HCl (pH = 7.5) and stained with DAPI (4′,6-diamino-2-phenylindole, Vectashield Mounting Medium H1200; Vector Laboratories, Burlingame, CA,USA). Slides were examined under an OlympusBX50 fluorescence photomicroscope equipped with an appropriate filter combination. The extent of DNA damage was quantified by the length of DNA migration, which was visually determined in 100 randomly selected and non overlapping cells. DNA damage was classified in four classes (0–I, undamaged; II, minimum damage; III, medium damage; IV, maximum damage), considering Cavaş and Könen (2007). Data are expressed as the mean number of damaged cells (sum of Classes II, III, and IV) and the mean comet score for each treatment group. The GDI was calculated for each test compound following Pitarque et al. (1999) using the formula GDI = [I(I) + 2(II) + 3(III) + 4(IV)/N(0–IV)], where 0–IV represents the nucleoid type, and N0–NIV represent the total number of nucleoids scored.
2.5 Statistical analysis
ANOVA one-way (analysis of variance) with post-hoc Dunnett test was performed to estimate the IMZT exposure- induced effects on body condition, organ index, liver enzymes activities (GST, CAT and AChE), frequencies of MN and others nuclear abnormalities, and GDI (response variables), at both exposure times evaluated (Zar, 2010). ANOVA assumptions were corroborated with Barlett test for the homogeneity of variances and χ2 test for normality. Then, data were logarithmic transformed to meet assumptions, in those cases that did not meet the assumptions of normality, a Kruskal–Wallis test was performed (Zar, 2010).
A principal component analysis (PCA) was performed considering each exposure scenario as a groping variable to improves interpretation of the results and allows obtain integral information of the biomarkers responses (or holistic vision). The integration of all biomarkers was made by using Components Principal Regression Analysis (Jackson, 1993; Jolliffe and Cadima, 2016). In addition, the relationship biomarkers and IMZT was evaluated with a correlation matrix (Pearson product moment correlation coefficient) by using simple linear regression. Tests of significance of the regression and correlation coefficients were performed following Zar (2010). The level of significance chosen was α = 0.05 for all tests, unless indicated otherwise. Analyses were performed using the R software 6 v. 2.11.1 (R Core Team 2010).